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Xiao Y, Ma J, Yuan X, Wang H, Ma F, Wu J, Chen Q, Hu J, Wang L, Zhang Z, Wang C, Li J, Wang W, Li B. Acid-Triggered Dual-Functional Hydrogel Platform for Enhanced Bone Regeneration. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2025; 12:e2415772. [PMID: 39868910 PMCID: PMC11923904 DOI: 10.1002/advs.202415772] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/27/2024] [Revised: 01/14/2025] [Indexed: 01/28/2025]
Abstract
Stem cell implantation holds promise for enhancing bone repair, but risks of pathogen transmission and malignant cell transformation should not be ignored. Compared to stem cell implantation, recruitment of endogenous stem cells to injured sites is more critical for in situ bone regeneration. In this study, based on the acidic microenvironment of bone injury, an HG-AA1:1-SDF-1α composite hydrogel with a dual-control intelligent switch function is developed by incorporating stromal cell-derived factor (SDF-1α), arginine carbon dots (Arg-CDs), and calcium ions (Ca2+) into the oxidized hyaluronic acid/gelatin methacryloyl (HG) hydrogel. The acidic microenvironment triggers the first switch (Schiff base bond is broken between HG-AA1:1 and SDF-1α) of HG-AA1:1-SDF-1α composite hydrogel to continuously release SDF-1α. Compared to the neutral (pH 7.4) media, the cumulative release of SDF-1α in acidic (pH 5.5) media is ≈2.5 times higher, which enhances the migration and recruitment of endogenous mesenchymal stem cells (MSCs). The recruited MSCs immediately initiate the second switch and metabolize Arg-CDs into the bioactive nitric oxide (NO) in the presence of Ca2+, activating NO/cyclic guanosine monophosphate (cGMP) signaling pathway to promote angiogenesis. Therefore, the engineered HG-AA1:1-SDF-1α composite hydrogel shows promising potential to achieve "coupling osteogenesis and angiogenesis" for bone regeneration.
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Affiliation(s)
- Yao Xiao
- The First Affiliated Hospital of Shihezi UniversityShiheziXinjiang832000China
| | - Jinjin Ma
- Medical 3D Printing CenterOrthopedic InstituteDepartment of Orthopedic SurgeryThe First Affiliated HospitalSchool of Basic Medical SciencesMOE Key Laboratory of Geriatric Diseases and ImmunologySuzhou Medical CollegeSoochow UniversitySuzhouJiangsu215000China
| | - Xiaonan Yuan
- Medical 3D Printing CenterOrthopedic InstituteDepartment of Orthopedic SurgeryThe First Affiliated HospitalSchool of Basic Medical SciencesMOE Key Laboratory of Geriatric Diseases and ImmunologySuzhou Medical CollegeSoochow UniversitySuzhouJiangsu215000China
| | - Huan Wang
- Medical 3D Printing CenterOrthopedic InstituteDepartment of Orthopedic SurgeryThe First Affiliated HospitalSchool of Basic Medical SciencesMOE Key Laboratory of Geriatric Diseases and ImmunologySuzhou Medical CollegeSoochow UniversitySuzhouJiangsu215000China
| | - Fengyu Ma
- Medical 3D Printing CenterOrthopedic InstituteDepartment of Orthopedic SurgeryThe First Affiliated HospitalSchool of Basic Medical SciencesMOE Key Laboratory of Geriatric Diseases and ImmunologySuzhou Medical CollegeSoochow UniversitySuzhouJiangsu215000China
| | - Jun Wu
- Medical 3D Printing CenterOrthopedic InstituteDepartment of Orthopedic SurgeryThe First Affiliated HospitalSchool of Basic Medical SciencesMOE Key Laboratory of Geriatric Diseases and ImmunologySuzhou Medical CollegeSoochow UniversitySuzhouJiangsu215000China
| | - Qianglong Chen
- Medical 3D Printing CenterOrthopedic InstituteDepartment of Orthopedic SurgeryThe First Affiliated HospitalSchool of Basic Medical SciencesMOE Key Laboratory of Geriatric Diseases and ImmunologySuzhou Medical CollegeSoochow UniversitySuzhouJiangsu215000China
| | - Jie Hu
- Medical 3D Printing CenterOrthopedic InstituteDepartment of Orthopedic SurgeryThe First Affiliated HospitalSchool of Basic Medical SciencesMOE Key Laboratory of Geriatric Diseases and ImmunologySuzhou Medical CollegeSoochow UniversitySuzhouJiangsu215000China
| | - Lijie Wang
- Sanitation & Environment Technology Institute of Soochow UniversitySuzhouJiangsu215163China
| | - Zhendong Zhang
- The First Affiliated Hospital of Shihezi UniversityShiheziXinjiang832000China
| | - Chao Wang
- The First Affiliated Hospital of Shihezi UniversityShiheziXinjiang832000China
| | - Jiaying Li
- Medical 3D Printing CenterOrthopedic InstituteDepartment of Orthopedic SurgeryThe First Affiliated HospitalSchool of Basic Medical SciencesMOE Key Laboratory of Geriatric Diseases and ImmunologySuzhou Medical CollegeSoochow UniversitySuzhouJiangsu215000China
| | - Weishan Wang
- The First Affiliated Hospital of Shihezi UniversityShiheziXinjiang832000China
| | - Bin Li
- Medical 3D Printing CenterOrthopedic InstituteDepartment of Orthopedic SurgeryThe First Affiliated HospitalSchool of Basic Medical SciencesMOE Key Laboratory of Geriatric Diseases and ImmunologySuzhou Medical CollegeSoochow UniversitySuzhouJiangsu215000China
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Bell JA, Mayfield CK, Collon K, Chang S, Gallo MC, Lechtholz-Zey E, Ayad M, Sugiyam O, Tang AH, Park SH, Lieberman JR. In vivo effects of cell seeding technique in an ex vivo regional gene therapy model for bone regeneration. J Biomed Mater Res A 2024; 112:1688-1698. [PMID: 38602243 DOI: 10.1002/jbm.a.37718] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2023] [Revised: 03/14/2024] [Accepted: 03/25/2024] [Indexed: 04/12/2024]
Abstract
When delivering cells on a scaffold to treat a bone defect, the cell seeding technique determines the number and distribution of cells within a scaffold, however the optimal technique has not been established. This study investigated if human adipose-derived stem cells (ASCs) transduced with a lentiviral vector to overexpress bone morphogenetic protein 2 (BMP-2) and loaded on a scaffold using dynamic orbital shaker could reduce the total cell dose required to heal a critical sized bone defect when compared with static seeding. Human ASCs were loaded onto a collagen/biphasic ceramic scaffold using static loading and dynamic orbital shaker techniques, compared with our labs standard loading technique, and implanted into femoral defects of nude rats. Both a low dose and standard dose of transduced cells were evaluated. Outcomes investigated included BMP-2 production, radiographic healing, micro-computerized tomography, histologic assessment, and biomechanical torsional testing. BMP-2 production was higher in the orbital shaker cohort compared with the static seeding cohort. No statistically significant differences were noted in radiographic, histomorphometric, and biomechanical outcomes between the low-dose static and dynamic seeding groups, however the standard-dose static seeding cohort had superior biomechanical properties. The standard-dose 5 million cell dose standard loading cohort had superior maximum torque and torsional stiffness on biomechanical testing. The use of orbital shaker technique was labor intensive and did not provide equivalent biomechanical results with the use of fewer cells.
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Affiliation(s)
- Jennifer A Bell
- Department of Orthopaedic Surgery, Keck School of Medicine of USC, Los Angeles, California, USA
| | - Cory K Mayfield
- Department of Orthopaedic Surgery, Keck School of Medicine of USC, Los Angeles, California, USA
| | - Kevin Collon
- Department of Orthopaedic Surgery, Keck School of Medicine of USC, Los Angeles, California, USA
| | - Stephanie Chang
- Department of Orthopaedic Surgery, Keck School of Medicine of USC, Los Angeles, California, USA
| | - Matthew C Gallo
- Department of Orthopaedic Surgery, Keck School of Medicine of USC, Los Angeles, California, USA
| | - Elizabeth Lechtholz-Zey
- Department of Orthopaedic Surgery, Keck School of Medicine of USC, Los Angeles, California, USA
| | - Mina Ayad
- Department of Orthopaedic Surgery, Keck School of Medicine of USC, Los Angeles, California, USA
| | - Osamu Sugiyam
- Department of Orthopaedic Surgery, Keck School of Medicine of USC, Los Angeles, California, USA
| | - Amy H Tang
- Department of Orthopaedic Surgery, Keck School of Medicine of USC, Los Angeles, California, USA
| | - Sang-Hyun Park
- J. Vernon Luck Orthopaedic Research Center, Orthopaedic Institute for Children, Los Angeles, California, USA
| | - Jay R Lieberman
- Department of Orthopaedic Surgery, Keck School of Medicine of USC, Los Angeles, California, USA
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Murayama M, Chow SK, Lee ML, Young B, Ergul YS, Shinohara I, Susuki Y, Toya M, Gao Q, Goodman SB. The interactions of macrophages, lymphocytes, and mesenchymal stem cells during bone regeneration. Bone Joint Res 2024; 13:462-473. [PMID: 39237112 PMCID: PMC11377107 DOI: 10.1302/2046-3758.139.bjr-2024-0122.r1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 09/07/2024] Open
Abstract
Bone regeneration and repair are crucial to ambulation and quality of life. Factors such as poor general health, serious medical comorbidities, chronic inflammation, and ageing can lead to delayed healing and nonunion of fractures, and persistent bone defects. Bioengineering strategies to heal bone often involve grafting of autologous bone marrow aspirate concentrate (BMAC) or mesenchymal stem cells (MSCs) with biocompatible scaffolds. While BMAC shows promise, variability in its efficacy exists due to discrepancies in MSC concentration and robustness, and immune cell composition. Understanding the mechanisms by which macrophages and lymphocytes - the main cellular components in BMAC - interact with MSCs could suggest novel strategies to enhance bone healing. Macrophages are polarized into pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes, and influence cell metabolism and tissue regeneration via the secretion of cytokines and other factors. T cells, especially helper T1 (Th1) and Th17, promote inflammation and osteoclastogenesis, whereas Th2 and regulatory T (Treg) cells have anti-inflammatory pro-reconstructive effects, thereby supporting osteogenesis. Crosstalk among macrophages, T cells, and MSCs affects the bone microenvironment and regulates the local immune response. Manipulating the proportion and interactions of these cells presents an opportunity to alter the local regenerative capacity of bone, which potentially could enhance clinical outcomes.
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Affiliation(s)
- Masatoshi Murayama
- Department of Orthopaedic Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Simon K. Chow
- Department of Orthopaedic Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Max L. Lee
- Department of Orthopaedic Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Bill Young
- Department of Orthopaedic Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Yasemin S. Ergul
- Department of Orthopaedic Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Issei Shinohara
- Department of Orthopaedic Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Yosuke Susuki
- Department of Orthopaedic Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Masakazu Toya
- Department of Orthopaedic Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Qi Gao
- Department of Orthopaedic Surgery, Stanford University School of Medicine, Stanford, California, USA
| | - Stuart B. Goodman
- Department of Orthopaedic Surgery, Stanford University School of Medicine, Stanford, California, USA
- Department of Bioengineering, Stanford University School of Medicine, Stanford, California, USA
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Haskell A, White BP, Rogers RE, Goebel E, Lopez MG, Syvyk AE, de Oliveira DA, Barreda HA, Benton J, Benavides OR, Dalal S, Bae E, Zhang Y, Maitland K, Nikolov Z, Liu F, Lee RH, Kaunas R, Gregory CA. Scalable manufacture of therapeutic mesenchymal stromal cell products on customizable microcarriers in vertical wheel bioreactors that improve direct visualization, product harvest, and cost. Cytotherapy 2024; 26:372-382. [PMID: 38363250 PMCID: PMC11057043 DOI: 10.1016/j.jcyt.2024.01.009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2023] [Revised: 01/23/2024] [Accepted: 01/27/2024] [Indexed: 02/17/2024]
Abstract
BACKGROUND AIMS Human mesenchymal stromal cells (hMSCs) and their secreted products show great promise for treatment of musculoskeletal injury and inflammatory or immune diseases. However, the path to clinical utilization is hampered by donor-tissue variation and the inability to manufacture clinically relevant yields of cells or their products in a cost-effective manner. Previously we described a method to produce chemically and mechanically customizable gelatin methacryloyl (GelMA) microcarriers for culture of hMSCs. Herein, we demonstrate scalable GelMA microcarrier-mediated expansion of induced pluripotent stem cell (iPSC)-derived hMSCs (ihMSCs) in 500 mL and 3L vertical wheel bioreactors, offering several advantages over conventional microcarrier and monolayer-based expansion strategies. METHODS Human mesenchymal stromal cells derived from induced pluripotent cells were cultured on custom-made spherical gelatin methacryloyl microcarriers in single-use vertical wheel bioreactors (PBS Biotech). Cell-laden microcarriers were visualized using confocal microscopy and elastic light scattering methodologies. Cells were assayed for viability and differentiation potential in vitro by standard methods. Osteogenic cell matrix derived from cells was tested in vitro for osteogenic healing using a rodent calvarial defect assay. Immune modulation was assayed with an in vivo peritonitis model using Zymozan A. RESULTS The optical properties of GelMA microcarriers permit noninvasive visualization of cells with elastic light scattering modalities, and harvest of product is streamlined by microcarrier digestion. At volumes above 500 mL, the process is significantly more cost-effective than monolayer culture. Osteogenic cell matrix derived from ihMSCs expanded on GelMA microcarriers exhibited enhanced in vivo bone regenerative capacity when compared to bone morphogenic protein 2, and the ihMSCs exhibited superior immunosuppressive properties in vivo when compared to monolayer-generated ihMSCs. CONCLUSIONS These results indicate that the cell expansion strategy described here represents a superior approach for efficient generation, monitoring and harvest of therapeutic MSCs and their products.
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Affiliation(s)
- Andrew Haskell
- Department of Cell Biology and Genetics, Texas A&M School of Medicine, Bryan, Texas, USA
| | - Berkley P White
- Department of Biomedical Engineering, Texas A&M University, College Station, Texas, USA
| | - Robert E Rogers
- Department of Cell Biology and Genetics, Texas A&M School of Medicine, Bryan, Texas, USA
| | - Erin Goebel
- Department of Cell Biology and Genetics, Texas A&M School of Medicine, Bryan, Texas, USA; Department of Biomedical Engineering, Texas A&M University, College Station, Texas, USA
| | - Megan G Lopez
- Department of Cell Biology and Genetics, Texas A&M School of Medicine, Bryan, Texas, USA
| | - Andrew E Syvyk
- National Center for Therapeutics Manufacturing, Texas A&M University, College Station, Texas, USA
| | - Daniela A de Oliveira
- National Center for Therapeutics Manufacturing, Texas A&M University, College Station, Texas, USA; Biological and Agricultural Engineering, Texas A&M University, College Station, Texas, USA
| | - Heather A Barreda
- Department of Cell Biology and Genetics, Texas A&M School of Medicine, Bryan, Texas, USA
| | - Joshua Benton
- Department of Cell Biology and Genetics, Texas A&M School of Medicine, Bryan, Texas, USA
| | - Oscar R Benavides
- Department of Biomedical Engineering, Texas A&M University, College Station, Texas, USA
| | - Sujata Dalal
- Department of Cell Biology and Genetics, Texas A&M School of Medicine, Bryan, Texas, USA
| | - EunHye Bae
- Department of Cell Biology and Genetics, Texas A&M School of Medicine, Bryan, Texas, USA
| | - Yu Zhang
- Department of Cell Biology and Genetics, Texas A&M School of Medicine, Bryan, Texas, USA
| | - Kristen Maitland
- Department of Biomedical Engineering, Texas A&M University, College Station, Texas, USA; Imaging Program, Chan Zuckerberg Initiative, Redwood City, California, USA
| | - Zivko Nikolov
- National Center for Therapeutics Manufacturing, Texas A&M University, College Station, Texas, USA; Biological and Agricultural Engineering, Texas A&M University, College Station, Texas, USA
| | - Fei Liu
- Department of Cell Biology and Genetics, Texas A&M School of Medicine, Bryan, Texas, USA
| | - Ryang Hwa Lee
- Department of Cell Biology and Genetics, Texas A&M School of Medicine, Bryan, Texas, USA
| | - Roland Kaunas
- Department of Biomedical Engineering, Texas A&M University, College Station, Texas, USA.
| | - Carl A Gregory
- Department of Cell Biology and Genetics, Texas A&M School of Medicine, Bryan, Texas, USA.
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Mayfield CK, Lechtholz-Zey E, Ayad M, Sugiyama O, Lieberman JR. Human Bone Marrow versus Adipose-Derived Stem Cells: Influence of Donor Characteristics on Expandability and Implications for Osteogenic Ex Vivo BMP-2 Regional Gene Therapy. J Tissue Eng Regen Med 2023; 2023:8061890. [PMID: 40226412 PMCID: PMC11919156 DOI: 10.1155/2023/8061890] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2023] [Revised: 08/01/2023] [Accepted: 08/04/2023] [Indexed: 04/15/2025]
Abstract
Novel treatment strategies for segmental bone loss in orthopaedic surgery remain under investigation. Regional gene therapy that involves transduction of mesenchymal stem cells with a lentiviral vector that expresses BMP-2 has gained particular interest as this strategy provides osteogenic and osteoinductive factors for bone growth. In particular, transduced adipose-derived stems cells (ASCs) and bone marrow-derived stem cells (BMSCs) have emerged as the leading candidates for the treatment of segmental defects in preclinical models. The aim of the present study was to evaluate the influence of demographic information on in vitro growth characteristics and bone morphogenetic protein-2 production following lentiviral transduction in a large cohort of human donors. We further sought to assess the effects of ASC harvest site on cell yield and growth characteristics. We evaluated a total of 187 human donors (124 adipose harvests and 63 bone marrow aspirates) in our cohort. We found that across all donors, ASCs demonstrated favorable growth characteristics and could be cultured in vitro more reliably than BMSCs regardless of patient-related factors. Furthermore, we noted that following lentiviral transduction, ASCs produced significantly higher levels of BMP-2 compared to BMSCs. Lastly, despite higher initial cell yields from lipoaspirate, posttransduction BMP-2 production was less than that of infrapatellar fat pad samples. These results support the continued investigation of ASCs as a cellular delivery vehicle for regional gene therapy to deliver osteoinductive proteins to specific anatomic bone repair sites.
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Affiliation(s)
- Cory K. Mayfield
- Keck School of Medicine of USC, Department of Orthopaedic Surgery, Los Angeles, USA
| | | | - Mina Ayad
- Keck School of Medicine of USC, Department of Orthopaedic Surgery, Los Angeles, USA
| | - Osamu Sugiyama
- Keck School of Medicine of USC, Department of Orthopaedic Surgery, Los Angeles, USA
| | - Jay R. Lieberman
- Keck School of Medicine of USC, Department of Orthopaedic Surgery, Los Angeles, USA
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Collon K, Bell JA, Gallo MC, Chang SW, Bougioukli S, Sugiyama O, Tassey J, Hollis R, Heckmann N, Oakes DA, Longjohn DB, Evseenko D, Kohn DB, Lieberman JR. Influence of donor age and comorbidities on transduced human adipose-derived stem cell in vitro osteogenic potential. Gene Ther 2022; 30:369-376. [PMID: 36216880 PMCID: PMC10086075 DOI: 10.1038/s41434-022-00367-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2022] [Revised: 09/24/2022] [Accepted: 09/27/2022] [Indexed: 01/17/2023]
Abstract
Human adipose-derived mesenchymal stem cells (ASCs) transduced with a lentiviral vector system to express bone morphogenetic protein 2 (LV-BMP-2) have been shown to reliably heal bone defects in animal models. However, the influence of donor characteristics such as age, sex, race, and medical co-morbidities on ASC yield, growth and bone regenerative capacity, while critical to the successful clinical translation of stem cell-based therapies, are not well understood. Human ASCs isolated from the infrapatellar fat pads in 122 ASC donors were evaluated for cell growth characteristics; 44 underwent additional analyses to evaluate in vitro osteogenic potential, with and without LV-BMP-2 transduction. We found that while female donors demonstrated significantly higher cell yield and ASC growth rates, age, race, and the presence of co-morbid conditions were not associated with differences in proliferation. Donor demographics or the presence of comorbidities were not associated with differences in in vitro osteogenic potential or stem cell differentiation, except that transduced ASCs from healthy donors produced more BMP-2 at day 2. Overall, donor age, sex, race, and the presence of co-morbid conditions had a limited influence on cell yield, proliferation, self-renewal capacity, and osteogenic potential for non-transduced and transduced (LV-BMP-2) ASCs. These results suggest that ASCs are a promising resource for both autologous and allogeneic cell-based gene therapy applications.
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Affiliation(s)
- Kevin Collon
- Department of Orthopaedic Surgery, Keck School of Medicine of the University of Southern California, 2011 Zonal Ave,HMR 702, Los Angeles, CA, 90089, USA.
| | - Jennifer A Bell
- Department of Orthopaedic Surgery, Keck School of Medicine of the University of Southern California, 2011 Zonal Ave,HMR 702, Los Angeles, CA, 90089, USA
| | - Matthew C Gallo
- Department of Orthopaedic Surgery, Keck School of Medicine of the University of Southern California, 2011 Zonal Ave,HMR 702, Los Angeles, CA, 90089, USA
| | - Stephanie W Chang
- Department of Orthopaedic Surgery, Keck School of Medicine of the University of Southern California, 2011 Zonal Ave,HMR 702, Los Angeles, CA, 90089, USA
| | - Sofia Bougioukli
- Department of Orthopaedic Surgery, Keck School of Medicine of the University of Southern California, 2011 Zonal Ave,HMR 702, Los Angeles, CA, 90089, USA
| | - Osamu Sugiyama
- Department of Orthopaedic Surgery, Keck School of Medicine of the University of Southern California, 2011 Zonal Ave,HMR 702, Los Angeles, CA, 90089, USA
| | - Jade Tassey
- Department of Orthopaedic Surgery, Keck School of Medicine of the University of Southern California, 2011 Zonal Ave,HMR 702, Los Angeles, CA, 90089, USA
| | - Roger Hollis
- Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA, USA
| | - Nathanael Heckmann
- Department of Orthopaedic Surgery, Keck School of Medicine of the University of Southern California, 2011 Zonal Ave,HMR 702, Los Angeles, CA, 90089, USA
| | - Daniel A Oakes
- Department of Orthopaedic Surgery, Keck School of Medicine of the University of Southern California, 2011 Zonal Ave,HMR 702, Los Angeles, CA, 90089, USA
| | - Donald B Longjohn
- Department of Orthopaedic Surgery, Keck School of Medicine of the University of Southern California, 2011 Zonal Ave,HMR 702, Los Angeles, CA, 90089, USA
| | - Denis Evseenko
- Department of Orthopaedic Surgery, Keck School of Medicine of the University of Southern California, 2011 Zonal Ave,HMR 702, Los Angeles, CA, 90089, USA
| | - Donald B Kohn
- Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA, USA
| | - Jay R Lieberman
- Department of Orthopaedic Surgery, Keck School of Medicine of the University of Southern California, 2011 Zonal Ave,HMR 702, Los Angeles, CA, 90089, USA
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Dobson LK, Zeitouni S, McNeill EP, Bearden RN, Gregory CA, Saunders WB. Canine Mesenchymal Stromal Cell-Mediated Bone Regeneration is Enhanced in the Presence of Sub-Therapeutic Concentrations of BMP-2 in a Murine Calvarial Defect Model. Front Bioeng Biotechnol 2021; 9:764703. [PMID: 34796168 PMCID: PMC8592971 DOI: 10.3389/fbioe.2021.764703] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2021] [Accepted: 09/27/2021] [Indexed: 11/15/2022] Open
Abstract
Novel bone regeneration strategies often show promise in rodent models yet are unable to successfully translate to clinical therapy. Sheep, goats, and dogs are used as translational models in preparation for human clinical trials. While human MSCs (hMSCs) undergo osteogenesis in response to well-defined protocols, canine MSCs (cMSCs) are more incompletely characterized. Prior work suggests that cMSCs require additional agonists such as IGF-1, NELL-1, or BMP-2 to undergo robust osteogenic differentiation in vitro. When compared directly to hMSCs, cMSCs perform poorly in vivo. Thus, from both mechanistic and clinical perspectives, cMSC and hMSC-mediated bone regeneration may differ. The objectives of this study were twofold. The first was to determine if previous in vitro findings regarding cMSC osteogenesis were substantiated in vivo using an established murine calvarial defect model. The second was to assess in vitro ALP activity and endogenous BMP-2 gene expression in both canine and human MSCs. Calvarial defects (4 mm) were treated with cMSCs, sub-therapeutic BMP-2, or the combination of cMSCs and sub-therapeutic BMP-2. At 28 days, while there was increased healing in defects treated with cMSCs, defects treated with cMSCs and BMP-2 exhibited the greatest degree of bone healing as determined by quantitative μCT and histology. Using species-specific qPCR, cMSCs were not detected in relevant numbers 10 days after implantation, suggesting that bone healing was mediated by anabolic cMSC or ECM-driven cues and not via engraftment of cMSCs. In support of this finding, defects treated with cMSC + BMP-2 exhibited robust deposition of Collagens I, III, and VI using immunofluorescence. Importantly, cMSCs exhibited minimal ALP activity unless cultured in the presence of BMP-2 and did not express endogenous canine BMP-2 under any condition. In contrast, human MSCs exhibited robust ALP activity in all conditions and expressed human BMP-2 when cultured in control and osteoinduction media. This is the first in vivo study in support of previous in vitro findings regarding cMSC osteogenesis, namely that cMSCs require additional agonists to initiate robust osteogenesis. These findings are highly relevant to translational cell-based bone healing studies and represent an important finding for the field of canine MSC-mediated bone regeneration.
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Affiliation(s)
- Lauren K Dobson
- Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, United States
| | - Suzanne Zeitouni
- Department of Molecular and Cellular Medicine, Institute for Regenerative Medicine, Texas A&M Health Science Center, College Station, TX, United States
| | - Eoin P McNeill
- Department of Molecular and Cellular Medicine, Institute for Regenerative Medicine, Texas A&M Health Science Center, College Station, TX, United States
| | - Robert N Bearden
- Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, United States
| | - Carl A Gregory
- Department of Molecular and Cellular Medicine, Institute for Regenerative Medicine, Texas A&M Health Science Center, College Station, TX, United States
| | - W Brian Saunders
- Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, United States
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Cercone M, Greenfield MR, Fortier LA. Bone Marrow Concentrate Mesenchymal Stromal Cells Do not Correlate With Nucleated Cell Count or Colony Forming Units. JOURNAL OF CARTILAGE & JOINT PRESERVATION 2021; 1:100017. [DOI: 10.1016/j.jcjp.2021.100017] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2025]
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9
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Musculoskeletal tissue engineering: Regional gene therapy for bone repair. Biomaterials 2021; 275:120901. [PMID: 34091300 DOI: 10.1016/j.biomaterials.2021.120901] [Citation(s) in RCA: 34] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2021] [Revised: 04/24/2021] [Accepted: 05/14/2021] [Indexed: 02/07/2023]
Abstract
Bone loss associated with fracture nonunion, revision total joint arthroplasty (TJA), and pseudoarthrosis of the spine presents a challenging clinical scenario for the orthopaedic surgeon. Current treatment options including autograft, allograft, bone graft substitutes, and bone transport techniques are associated with significant morbidity, high costs, and prolonged treatment regimens. Unfortunately, these treatment strategies have proven insufficient to safely and consistently heal bone defects in the stringent biological environments often encountered in clinical cases of bone loss. The application of tissue engineering (TE) to musculoskeletal pathology has uncovered exciting potential treatment strategies for challenging bone loss scenarios in orthopaedic surgery. Regional gene therapy involves the local implantation of nucleic acids or genetically modified cells to direct specific protein expression, and has shown promise as a potential TE technique for the regeneration of bone. Preclinical studies in animal models have demonstrated the ability of regional gene therapy to safely and effectively heal critical sized bone defects which otherwise do not heal. The purpose of the present review is to provide a comprehensive overview of the current status of gene therapy applications for TE in challenging bone loss scenarios, with an emphasis on gene delivery methods and models, scaffold biomaterials, preclinical results, and future directions.
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10
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Barrientos FJ, Redondo LM, Alberca M, Sánchez AM, García-Sancho J. Bone regeneration with autologous adipose-derived mesenchymal stem cells: A reliable experimental model in rats. MethodsX 2020; 7:101137. [PMID: 33251125 PMCID: PMC7679249 DOI: 10.1016/j.mex.2020.101137] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2020] [Accepted: 11/05/2020] [Indexed: 01/14/2023] Open
Abstract
The adult mesenchymal stem cell (MSC) has been proposed to be the definitive tool in regenerative medicine due to its multi-differentiation potential and expansion capacity ex vivo. The use of MSCs on bone regeneration has been assessed in several studies, obtaining promising results. However, the endless combinations that can be tested and the heterogeneity in the experimental conditions become a drawback when comparing results between authors. Moreover, it is very hard to find autologous studies using adipose-derived MSCs (AD-MSC) in rodents, which is the most used preclinical animal model. In this article an experimental model for basic bone tissue engineering research is described and justified, on which adult AD-MSCs are safely isolated from the rat dorsal interscapular fat pad, allowing ex vivo expansion and autogenous orthotopic reimplantation in a bilateral mandibular bone defect made in the same animal. This reliable and reproducible model provides a simple way to perform basic experimentation studies in a small animal model using autologous MSC for bone regeneration or cell therapy techniques prior to improve the research on large animal models.
Predictable and safe harvest of adipose-derived MSC. No need of animal sacrifice. Allows for autologous studies with the most frequently used animal model: the rat. No need of allogeneic or human MSC use and, therefore, immunological concerns are avoided. Bilateral mandibular critical size defect to allow direct control/experimental comparison.
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Affiliation(s)
| | - Luis Miguel Redondo
- Servicio de Cirugía Maxilofacial, Hospital del Rio Hortega, Valladolid, Spain
| | - Mercedes Alberca
- Citospin SL, Edificio I+D Campus Miguel Delibes, Valladolid, Spain
| | - Ana María Sánchez
- Instituto de Biología y Genética Molecular (IBGM), Universidad de Valladolid y Centro Superior de Investigaciones Científicas (CSIC), Valladolid, Spain
| | - Javier García-Sancho
- Instituto de Biología y Genética Molecular (IBGM), Universidad de Valladolid y Centro Superior de Investigaciones Científicas (CSIC), Valladolid, Spain
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11
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Hikmawati D, Kulsum U, Rudyardjo DI, Apsari R. Biocompatibility and osteoconductivity of scaffold porous composite collagen–hydroxyapatite based coral for bone regeneration. OPEN CHEM 2020. [DOI: 10.1515/chem-2020-0080] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022] Open
Abstract
AbstractThe synthesis of collagen–hydroxyapatite composites has been carried out, and the biocompatibility and osteoconductivity properties have been tested. This research was conducted to determine the ability of hydroxyapatite–collagen composites to support the bone growth through the graft surface. Hydroxyapatite used in this study was synthesized from coral with a purity of 96.6%, while collagen was extracted from the chicken claw. The process of forming a scaffold of collagen–hydroxyapatite composites was carried out using the freeze-drying method at −80°C for 4 h. The biocompatibility characteristics of the sample through the cytotoxicity tests showed that the percentage of viable cells in collagen–hydroxyapatite biocomposite was 108.2%, which is higher than the percentage of viable cells of hydroxyapatite or collagen material. When the viable cell is above 100%, collagen–hydroxyapatite composites have excellent osteoconductivity as a material for bone regeneration.
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Affiliation(s)
| | - Dyah Hikmawati
- Department of Physics, Faculty of Science and Technology, Universitas Airlangga, Kampus C, Jalan Mulyorejo, Surabaya, East Java, 60116, Indonesia
| | - Umi Kulsum
- Biomedical Engineering Program Study, Department of Physics, Faculty of Science and Technology, Universitas Airlangga, Kampus C, Jalan Mulyorejo Surabaya, East Java, 60116, Indonesia
| | - Djony Izak Rudyardjo
- Department of Physics, Faculty of Science and Technology, Universitas Airlangga, Kampus C, Jalan Mulyorejo, Surabaya, East Java, 60116, Indonesia
| | - Retna Apsari
- Department of Physics, Faculty of Science and Technology, Universitas Airlangga, Kampus C, Jalan Mulyorejo, Surabaya, East Java, 60116, Indonesia
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12
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Shi L, Tee BC, Emam H, Prokes R, Larsen P, Sun Z. Enhancement of bone marrow aspirate concentrate with local self-healing corticotomies. Tissue Cell 2020; 66:101383. [PMID: 32933706 DOI: 10.1016/j.tice.2020.101383] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2019] [Revised: 05/11/2020] [Accepted: 05/12/2020] [Indexed: 01/08/2023]
Abstract
Bone marrow aspirate concentrate (BMAC) is a potentially useful biological product for bone regeneration. This study investigated whether BMAC can be enriched by local minor corticotomies. Five 4-month-old domestic pigs were used with each pig undergoing two minor corticotomies at one randomly-selected tibia. Two weeks after the operation, bone marrow was aspirated from both tibiae and processed into BMAC samples. The amount of mesenchymal stem cells (MSCs) and the concentration of several regenerative growth factors contained in BMAC, as well as the proliferative and osteogenic differentiation capacity of MSCs, were compared between the corticotomy and the control sides. Another four weeks later, healing of the corticotomies was evaluated by radiographic and histological methods. The results demonstrated that BMAC from the corticotomy side contained significantly more MSCs than the control side. MSCs from the corticotomy side also proliferated significantly faster and tended to have stronger osteogenic differentiation than those from the control side. In contrast, the protein concentration of TGF-β, BMP-2 and PDGF contained in BMAC was only minimally changed by the corticotomies. The corticotomies in all pigs healed uneventfully, showing complete obliteration of the corticotomy gaps on CT images. Comparison between the two sides showed that the corticotomy side had thicker and denser cortical bone and more abundant osteogenic cell differentiation than the control side. These findings suggest that the quantity and proliferative/osteogenic differentiation capacity of MSCs contained in local BMAC can be enhanced by minor corticotomies, and spontaneous healing of the corticotomy can be completed within 6 weeks of the operation.
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Affiliation(s)
- Lei Shi
- Department of Pediatric Dentistry, Ninth People's Hospital, Shanghai Jiaotong University, School of Medicine, Shanghai 200011, China; Division of Orthodontics, College of Dentistry, The Ohio State University, Rm 4088 Postle Hall, 305 W 12th Ave, 43210 Columbus, OH, USA
| | - Boon Ching Tee
- Division of Biosciences, College of Dentistry, The Ohio State University, Columbus, OH, USA
| | - Hany Emam
- Division of Oral and Maxillofacial Surgery, College of Dentistry, The Ohio State University, Columbus, OH, USA
| | - Rachael Prokes
- Division of Oral and Maxillofacial Surgery, College of Dentistry, The Ohio State University, Columbus, OH, USA
| | - Peter Larsen
- Division of Oral and Maxillofacial Surgery, College of Dentistry, The Ohio State University, Columbus, OH, USA
| | - Zongyang Sun
- Division of Orthodontics, College of Dentistry, The Ohio State University, Rm 4088 Postle Hall, 305 W 12th Ave, 43210 Columbus, OH, USA.
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Connective Tissue Progenitor Analysis of Bone Marrow Aspirate Concentrate Harvested From the Body of the Ilium During Arthroscopic Acetabular Labral Repair. Arthroscopy 2020; 36:1311-1320. [PMID: 31958539 DOI: 10.1016/j.arthro.2019.11.125] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/15/2019] [Revised: 11/24/2019] [Accepted: 11/24/2019] [Indexed: 02/02/2023]
Abstract
PURPOSE To evaluate the number and concentration of progenitors of the bone marrow aspirate (BMA) harvest from the body of the ilium in comparison with other established aspiration sites. METHODS The inclusion criteria consisted of primary hip arthroscopy for acetabular labral tear. BMA was performed by placing an aspiration needle into the body of ilium just proximal to the sourcil in 33 patients. The BMA was centrifuged and processed in the operating room, resulting in approximately 3 to 5 mL of bone marrow aspirate concentrate (BMAC). Samples of both BMA and BMAC sample were analyzed. RESULTS The cohort of 30 patients had a mean number of nucleated cells of 24.0 million nucleated cells/cc of BMA. The BMAC samples had a mean connective tissue progenitor (CTP) cell concentration of 879.3 stem cells/cc of BMAC, a mean CTP prevalence of 34.1 stem cells/million nucleated cells, and a mean number of days to form colonies of 2.97 days. All 4 metrics of CTP harvest did not vary significantly with age, body mass index, sex, or laterality. The nucleated cell count was significantly associated with both CTP prevalence, r2 = 0.287 (P = .002), and CTP concentration, r2 = 0.388 (P < .001). CONCLUSIONS BMAC harvested from the body of the ilium during concurrent hip arthroscopy is a technically and biologically feasible option. Furthermore, the harvest site was found to have a CTP concentration that is similar or exceeds other published harvest sites. Finally, BMAC processing and application to areas of articular cartilage wear was performed efficiently and safely with no increase in morbidity or complications. CLINICAL RELEVANCE The body of the ilium is a reliable and rich source of CTP cells. This study may assist orthopaedic surgeons interested in performing biologic augmentation during hip surgery in determining a harvest site.
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14
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Obeid BA. Implants and grafts used in fractures for early healing. JOURNAL OF ORTHOPAEDICS AND SPINE 2020. [DOI: 10.4103/joas.joas_45_19] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/04/2022] Open
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15
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Bougioukli S, Alluri R, Pannell W, Sugiyama O, Vega A, Tang A, Skorka T, Park SH, Oakes D, Lieberman JR. Ex vivo gene therapy using human bone marrow cells overexpressing BMP-2: "Next-day" gene therapy versus standard "two-step" approach. Bone 2019; 128:115032. [PMID: 31398502 PMCID: PMC6813891 DOI: 10.1016/j.bone.2019.08.005] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/25/2019] [Revised: 08/02/2019] [Accepted: 08/05/2019] [Indexed: 01/13/2023]
Abstract
Traditionally, ex vivo gene therapy involves a two-step approach, with culture expansion of cells prior to transduction and implantation. We have tried to simplify this strategy and eliminate the time and cost associated with culture expansion, by introducing "next-day" regional gene therapy using human bone marrow cells. The purpose of this study was to determine whether a lentiviral vector (LV) carrying the cDNA for BMP-2 can transduce freshly isolated human BM cells, leading to abundant BMP production and bone formation in vivo, and evaluate the in vivo osteoinductive potential of "next-day" gene therapy and the standard "two-step" tissue culture expansion approach. To this end, human bone marrow cells (HBMC) from patients undergoing total hip arthroplasty were harvested, transduced with a BMP-2-expressing LV either overnight ("next day" gene therapy; ND) or after culture expansion (cultured "two-step" approach; C) and then implanted into a rat critical-sized femoral defect. The animals were randomly assigned to one of the following groups: I; ND-HBMC transduced with LV-TSTA BMP-2, II; ND-HBMC transduced with LV-TSTA GFP, III; non-transduced ND-HBMC; IV; C-HBMC transduced with LV-TSTA BMP-2, V; C-HBMC transduced with LV-TSTA-GFP, VI; non-transduced C-HBMC. Treatment with either "next-day" or cultured HBMC demonstrated a significant increase in new bone formation compared with all negative control groups as seen in plain radiographs, microCT and histologic/histomorphometric analysis. At 12 weeks post-op, complete defect union on plain X-rays occurred in 7/14 animals in the ND-HBMC/BMP-2 group and 12/14 in the C-HBMC/BMP-2 treated rats. The two-step approach was associated with more consistent results, a higher union rate, and superiority with regards to all of the studied bone healing parameters. In this study we demonstrate proof of concept that BMP-2-transduced human bone marrow cells can be used to enhance bone healing in segmental bone defects, and that regional gene therapy using lentiviral transduction has the osteoinductive potential to heal large bone defects in clinical settings.
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Affiliation(s)
- Sofia Bougioukli
- Department of Orthopaedic Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
| | - Ram Alluri
- Department of Orthopaedic Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
| | - William Pannell
- Department of Orthopaedic Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
| | - Osamu Sugiyama
- Department of Orthopaedic Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
| | - Andrew Vega
- Department of Orthopaedic Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
| | - Amy Tang
- Department of Orthopaedic Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
| | | | - Sang Hyun Park
- Orthopaedic Institute for Children, J. Vernon Luck. Sr., Orthopaedic Research Center, Los Angeles, CA, USA
| | - Daniel Oakes
- Department of Orthopaedic Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
| | - Jay R Lieberman
- Department of Orthopaedic Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.
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Park S, Arai Y, Kim BJ, Bello A, Ashraf S, Park H, Park KS, Lee SH. Suppression of SPRY4 Promotes Osteogenic Differentiation and Bone Formation of Mesenchymal Stem Cell. Tissue Eng Part A 2019; 25:1646-1657. [PMID: 30982407 DOI: 10.1089/ten.tea.2019.0056] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023] Open
Abstract
The directed differentiation of human adipose-derived stem cells (hASCs) into different cell types has shown great therapeutic potential in treating various diseases. To maximize the therapeutic potentials, researchers have tried manipulating master transcriptional genes that promote efficient differentiation of mesenchymal stem cells (MSCs) such as the MAPK/ERK signaling pathway. Sprouty (SPRY) is a family of proteins that are known to inhibit the MAPK/ERK signaling pathway. Although the role of some SPRY isoforms in MSC differentiation is known, the function of SPRY4 isoform has not been fully elucidated. In the present study, the role of SPRY4 in the multilineage differentiation of hASCs has been elucidated. To investigate the role of SPRY4 in hASC differentiation and tissue regeneration, we performed a transient knockdown of SPRY expression via a small interfering RNA (siSPRY4). Western blot and quantitative polymerase chain reaction results revealed that the treatment of siSPRY4 before induction of differentiation had no significant effect on adipogenic, but reduced chondrogenic, differentiation of hASCs. Interestingly, SPRY4 transient knockdown had a significant effect on the osteogenic differentiation as indicated by the increased messenger RNA (mRNA) and protein expression of osteogenic markers such as alkaline phosphatase (ALP; 2.3-fold) and osteopontin (OPN; 3.5-fold) and increased calcium deposition measured via Alizarin red staining (3.3-fold). Moreover, in vivo tissue regeneration of siSPRY4-treated hASCs in ectopic bone formation and calvarial defect mouse models showed higher bone volume (5.24-fold) and trabecular number (4.59-fold) assessed via histological and microcomputed tomography analyses. We also determined that the enhanced osteogenic differentiation in SPRY4-treated hASCs was due to the induction of ERK1/2 phosphorylation. Taken together, our results suggest that the regulation of SPRY4 through MAPK signaling is a potentially critical aspect on the osteogenic differentiation of hASCs and for bone tissue regeneration, and thus, may be utilized as a potent technique in the development of effective bone therapeutics. Impact Statement This study tried to expand our current understanding of the osteogenic differentiation of mesenchymal stem cells. The transient downregulation of the SPRY4 expression via small interfering RNA (siRNA) showed significant enhancement of the osteogenic differentiation of adipose-derived stem cells via the induction of ERK 1/2 phosphorylation. This suggests the possible mechanism to maximize the potential of stem cell as therapeutics and has a great potential in treating various bone-related diseases.
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Affiliation(s)
- Sunghyun Park
- Department of Medical Biotechnology, Dongguk University, Goyang-si, Republic of Korea.,Department of Biomedical Science, CHA University, Seongnam-si, Republic of Korea
| | - Yoshie Arai
- Department of Medical Biotechnology, Dongguk University, Goyang-si, Republic of Korea
| | - Byoung Ju Kim
- Department of Medical Biotechnology, Dongguk University, Goyang-si, Republic of Korea
| | - Alvin Bello
- Department of Medical Biotechnology, Dongguk University, Goyang-si, Republic of Korea.,Department of Integrative Engineering, Chung-Ang University, Seoul, Republic of Korea
| | - Sajjad Ashraf
- Department of Biomedical Science, CHA University, Seongnam-si, Republic of Korea
| | - Hansoo Park
- Department of Integrative Engineering, Chung-Ang University, Seoul, Republic of Korea
| | - Kyung-Soon Park
- Department of Biomedical Science, CHA University, Seongnam-si, Republic of Korea
| | - Soo-Hong Lee
- Department of Medical Biotechnology, Dongguk University, Goyang-si, Republic of Korea
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17
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Bougioukli S, Saitta B, Sugiyama O, Tang AH, Elphingstone J, Evseenko D, Lieberman JR. Lentiviral Gene Therapy for Bone Repair Using Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells. Hum Gene Ther 2019; 30:906-917. [PMID: 30773946 DOI: 10.1089/hum.2018.054] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Umbilical cord blood (UCB) has been increasingly explored as an alternative source of stem cells for use in regenerative medicine due to several advantages over other stem-cell sources, including the need for less stringent human leukocyte antigen matching. Combined with an osteoinductive signal, UCB-derived mesenchymal stem cells (MSCs) could revolutionize the treatment of challenging bone defects. This study aimed to develop an ex vivo regional gene-therapy strategy using BMP-2-transduced allogeneic UCB-MSCs to promote bone repair. To this end, human UCB-MSCs were transduced with a lentiviral vector carrying the cDNA for BMP-2 (LV-BMP-2). In vitro assays to determine the UCB-MSC osteogenic potential and BMP-2 production were followed by in vivo implantation of LV-BMP-2-transduced UCB-MSCs in a mouse hind-limb muscle pouch. Non-transduced and LV-GFP-transduced UCB-MSCs were used as controls. Transduction with LV-BMP-2 was associated with abundant BMP-2 production and induction of osteogenic differentiation in vitro. Implantation of BMP-2-transduced UCB-MSCs led to robust heterotopic bone formation 4 weeks postoperatively, as seen on radiographs and histology. These results, along with the fact that UCB-MSCs can be easily collected with no donor-site morbidity and low immunogenicity, suggest that UCB might be a preferable allogeneic source of MSCs to develop an ex vivo gene-therapy approach to treat difficult bone-repair scenarios.
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Affiliation(s)
- Sofia Bougioukli
- Department of Orthopaedic Surgery, Keck School of Medicine, University of Southern California, Los Angeles, California
| | - Biagio Saitta
- Department of Orthopaedic Surgery, Keck School of Medicine, University of Southern California, Los Angeles, California
| | - Osamu Sugiyama
- Department of Orthopaedic Surgery, Keck School of Medicine, University of Southern California, Los Angeles, California
| | - Amy H Tang
- Department of Orthopaedic Surgery, Keck School of Medicine, University of Southern California, Los Angeles, California
| | - Joseph Elphingstone
- Department of Orthopaedic Surgery, Keck School of Medicine, University of Southern California, Los Angeles, California
| | - Denis Evseenko
- Department of Orthopaedic Surgery, Keck School of Medicine, University of Southern California, Los Angeles, California
| | - Jay R Lieberman
- Department of Orthopaedic Surgery, Keck School of Medicine, University of Southern California, Los Angeles, California
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Schäfer R, DeBaun MR, Fleck E, Centeno CJ, Kraft D, Leibacher J, Bieback K, Seifried E, Dragoo JL. Quantitation of progenitor cell populations and growth factors after bone marrow aspirate concentration. J Transl Med 2019; 17:115. [PMID: 30961655 PMCID: PMC6454687 DOI: 10.1186/s12967-019-1866-7] [Citation(s) in RCA: 52] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2019] [Accepted: 03/28/2019] [Indexed: 12/15/2022] Open
Abstract
Background The number of Mesenchymal Stem/Stromal Cells (MSCs) in the human bone marrow (BM) is small compared to other cell types. BM aspirate concentration (BMAC) may be used to increase numbers of MSCs, but the composition of MSC subpopulations and growth factors after processing are unknown. The purpose of this study was to assess the enrichment of stem/progenitor cells and growth factors in BM aspirate by two different commercial concentration devices versus standard BM aspiration. Methods 120 mL of BM was aspirated from the iliac crest of 10 male donors. Each sample was processed simultaneously by either Emcyte GenesisCS® (Emcyte) or Harvest SmartPReP2 BMAC (Harvest) devices and compared to untreated BM aspirate. Samples were analyzed with multicolor flow cytometry for cellular viability and expression of stem/progenitor cells markers. Stem/progenitor cell content was verified by quantification of colony forming unit-fibroblasts (CFU-F). Platelet, red blood cell and total nucleated cell (TNC) content were determined using an automated hematology analyzer. Growth factors contents were analyzed with protein quantification assays. Statistical analyses were performed by ANOVA analysis of variance followed by Tukey’s multiple comparison test or Wilcoxon matched-pairs signed rank test with p < 0.05 for significance. Results Cell viability after processing was approximately 90% in all groups. Compared to control, both devices significantly enriched TNCs and platelets, as well as the CD45−CD73+ and CD45−CD73+CD90+ cell populations. Further, Harvest significantly concentrated CD45−CD10+, CD45−CD29+, CD45−CD90+, CD45−CD105+, CD45−CD119+ cells, and CD45dimCD90+CD271+ MSCs, whereas Emcyte significantly enriched CD45dimCD44+CD271+ MSCs. BM concentration also increased the numbers of CFU-F, platelet-derived growth factor, vascular endothelial growth factor, macrophage colony-stimulating factor, interleukin-1b, VCAM-1 and total protein. Neither system concentrated red blood cells, hematopoietic stem cells or bone morphogenetic proteins. Conclusion This data could contribute to the development of BMAC quality control assays as both BMAC systems concentrated platelets, growth factors and non-hematopoietic stem cell subpopulations with distinct phenotypes without loss of cell viability when compared to unprocessed BM. Electronic supplementary material The online version of this article (10.1186/s12967-019-1866-7) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Richard Schäfer
- Institute for Transfusion Medicine and Immunohematology, German Red Cross Blood Service Baden-Württemberg - Hessen gGmbH, Frankfurt Am Main, Germany
| | - Malcolm R DeBaun
- Department of Orthopedic Surgery, Stanford University School of Medicine, 450 Broadway, Redwood City, CA, 94063, USA
| | - Erika Fleck
- Institute for Transfusion Medicine and Immunohematology, German Red Cross Blood Service Baden-Württemberg - Hessen gGmbH, Frankfurt Am Main, Germany
| | | | - Daniela Kraft
- Institute for Transfusion Medicine and Immunohematology, German Red Cross Blood Service Baden-Württemberg - Hessen gGmbH, Frankfurt Am Main, Germany
| | - Johannes Leibacher
- Institute for Transfusion Medicine and Immunohematology, German Red Cross Blood Service Baden-Württemberg - Hessen gGmbH, Frankfurt Am Main, Germany
| | - Karen Bieback
- Institute of Transfusion Medicine and Immunology, Medical Faculty Mannheim, Heidelberg University, German Red Cross Blood Service Baden-Württemberg - Hessen gGmbH, Mannheim, Germany
| | - Erhard Seifried
- Institute for Transfusion Medicine and Immunohematology, German Red Cross Blood Service Baden-Württemberg - Hessen gGmbH, Frankfurt Am Main, Germany
| | - Jason L Dragoo
- Department of Orthopedic Surgery, Stanford University School of Medicine, 450 Broadway, Redwood City, CA, 94063, USA.
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González-Gil AB, Lamo-Espinosa JM, Muiños-López E, Ripalda-Cemboráin P, Abizanda G, Valdés-Fernández J, López-Martínez T, Flandes-Iparraguirre M, Andreu I, Elizalde MR, Stuckensen K, Groll J, De-Juan-Pardo EM, Prósper F, Granero-Moltó F. Periosteum-derived mesenchymal progenitor cells in engineered implants promote fracture healing in a critical-size defect rat model. J Tissue Eng Regen Med 2019; 13:742-752. [PMID: 30785671 DOI: 10.1002/term.2821] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2017] [Revised: 02/01/2019] [Accepted: 02/13/2019] [Indexed: 11/06/2022]
Abstract
An attractive alternative to bone autografts is the use of autologous mesenchymal progenitor cells (MSCs) in combination with biomaterials. We compared the therapeutic potential of different sources of mesenchymal stem cells in combination with biomaterials in a bone nonunion model. A critical-size defect was created in Sprague-Dawley rats. Animals were divided into six groups, depending on the treatment to be applied: bone defect was left empty (CTL); treated with live bone allograft (LBA); hrBMP-2 in collagen scaffold (CSBMP2 ); acellular polycaprolactone scaffold (PCL group); PCL scaffold containing periosteum-derived MSCs (PCLPMSCs ) and PCL containing bone marrow-derived MSCs (PCLBMSCs ). To facilitate cell tracking, both MSCs and bone graft were isolated from green fluorescent protein (GFP)-transgenic rats. CTL group did not show any signs of healing during the radiological follow-up (n = 6). In the LBA group, all the animals showed bone bridging (n = 6) whereas in the CSBMP2 group, four out of six animals demonstrated healing. In PCL and PCLPMSCs groups, a reduced number of animals showed radiological healing, whereas no healing was detected in the PCLBMSCs group. Using microcomputed tomography, the bone volume filling the defect was quantified, showing significant new bone formation in the LBA, CSBMP2 , and PCLPMSCs groups when compared with the CTL group. At 10 weeks, GFP positive cells were detected only in the LBA group and restricted to the outer cortical bone in close contact with the periosteum. Tracking of cellular implants demonstrated significant survival of the PMSCs when compared with BMSCs. In conclusion, PMSCs improve bone regeneration being suitable for mimetic autograft design.
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Affiliation(s)
- Ana B González-Gil
- Orthopaedic Surgery and Traumatology Department, Clínica Universidad de Navarra, Pamplona, Spain
| | - José M Lamo-Espinosa
- Orthopaedic Surgery and Traumatology Department, Clínica Universidad de Navarra, Pamplona, Spain
| | - Emma Muiños-López
- Cell Therapy Area, Centro de Investigación Médica Aplicada, IDISNA, Universidad de Navarra, Pamplona, Spain
| | | | - Gloria Abizanda
- Cell Therapy Area, Centro de Investigación Médica Aplicada, IDISNA, Universidad de Navarra, Pamplona, Spain
| | - José Valdés-Fernández
- Cell Therapy Area, Centro de Investigación Médica Aplicada, IDISNA, Universidad de Navarra, Pamplona, Spain
| | - Tania López-Martínez
- Cell Therapy Area, Centro de Investigación Médica Aplicada, IDISNA, Universidad de Navarra, Pamplona, Spain
| | | | - Ion Andreu
- TECNUN, Universidad de Navarra, San Sebastian, Spain
| | - María Reyes Elizalde
- TECNUN, Universidad de Navarra, San Sebastian, Spain.,CEIT, San Sebastian, Spain
| | - Kai Stuckensen
- Department of Functional Materials in Medicine and Dentistry, University of Würzburg, Würzburg, Germany
| | - Jürgen Groll
- Department of Functional Materials in Medicine and Dentistry, University of Würzburg, Würzburg, Germany
| | - Elena M De-Juan-Pardo
- Centre in Regenerative Medicine, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia
| | - Felipe Prósper
- Orthopaedic Surgery and Traumatology Department, Clínica Universidad de Navarra, Pamplona, Spain.,Cell Therapy Area, Centro de Investigación Médica Aplicada, IDISNA, Universidad de Navarra, Pamplona, Spain.,Hematology and Cell Therapy Area, Clínica Universidad de Navarra, Pamplona, Spain
| | - Froilán Granero-Moltó
- Orthopaedic Surgery and Traumatology Department, Clínica Universidad de Navarra, Pamplona, Spain.,Cell Therapy Area, Centro de Investigación Médica Aplicada, IDISNA, Universidad de Navarra, Pamplona, Spain
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Kurzyk A, Dębski T, Święszkowski W, Pojda Z. Comparison of adipose stem cells sources from various locations of rat body for their application for seeding on polymer scaffolds. JOURNAL OF BIOMATERIALS SCIENCE-POLYMER EDITION 2019; 30:376-397. [DOI: 10.1080/09205063.2019.1570433] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Affiliation(s)
- Agata Kurzyk
- Department of Regenerative Medicine, Maria Sklodowska Curie Institute – Oncology Center, Warsaw, Poland
| | - Tomasz Dębski
- Department of Regenerative Medicine, Maria Sklodowska Curie Institute – Oncology Center, Warsaw, Poland
| | - Wojciech Święszkowski
- Materials Design Division, Faculty of Material Science and Engineering, Warsaw University of Technology, Warsaw, Poland
| | - Zygmunt Pojda
- Department of Regenerative Medicine, Maria Sklodowska Curie Institute – Oncology Center, Warsaw, Poland
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Multiple integrin ligands provide a highly adhesive and osteoinductive surface that improves selective cell retention technology. Acta Biomater 2019; 85:106-116. [PMID: 30557698 DOI: 10.1016/j.actbio.2018.12.018] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2018] [Revised: 12/11/2018] [Accepted: 12/13/2018] [Indexed: 01/01/2023]
Abstract
Among various bone tissue engineering strategies, selective cell retention (SCR) technology has been used as a practical clinical method for bone graft manufacturing in real time. The more mesenchymal stem cells (MSCs) are retained, the better the osteoinductive microenvironment provided by the scaffold, which in turn promotes the osteogenesis of the SCR-fabricated bone grafts. Integrin receptors are crucial to cell-matrix adhesion and signal transduction. We designed a collagen-binding domain (CBD)-containing IKVAV-cRGD peptide (CBD-IKVAV-cRGD peptide) to complement the collagen-based demineralized bone matrix (DBM) with a functionalized surface containing multiple integrin ligands, which correspond to the highly expressed integrin subtypes on MSCs. This DBM/CBD-IKVAV-cRGD composite exhibited superior in vitro adhesion capacity to cultured MSCs, as determined by oscillatory cell adhesion assay, centrifugal cell adhesion assay and mimetic SCR. Moreover, it promoted the retention of MSC-like CD271+ cells and MSC-like CD90+/CD105+ cells in the clinical SCR method. Furthermore, the DBM/CBD-IKVAV-cRGD composite induced robust MSC osteogenesis, coupled with the activation of the downstream FAK-ERK1/2 signaling pathway of integrins. The SCR-prepared DBM/CBD-IKVAV-cRGD composite displayed superior in vivo osteogenesis, indicating that it may be potentially utilized as a biomaterial in SCR-mediated bone transplantation. STATEMENT OF SIGNIFICANCE: Selective cell retention technology (SCR) has been utilized in clinical settings to manufacture bioactive bone grafts. Specifically, demineralized bone matrix (DBM) is a widely-used SCR clinical biomaterial but it displays poor adhesion performance and osteoinduction. Improvements of the DBM that promote cell adhesion and osteoinduction will benefit SCR-prepared implants. In this work, we developed a novel peptide that complements the DBM with a functionalized surface of multiple integrin ligands, which are corresponding to integrin subtypes available on human bone marrow-derived mesenchymal stem cells (MSCs). Our results indicate this novel functionalized bioscaffold greatly increases SCR-mediated MSC adhesion and in vivo osteogenesis. Overall, this novel material has promising SCR applications and may likely provide highly bioactive bone implants in clinical settings.
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Ortved KF. Regenerative Medicine and Rehabilitation for Tendinous and Ligamentous Injuries in Sport Horses. Vet Clin North Am Equine Pract 2018; 34:359-373. [DOI: 10.1016/j.cveq.2018.04.012] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
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Oryan A, Baghaban Eslaminejad M, Kamali A, Hosseini S, Moshiri A, Baharvand H. RETRACTED ARTICLE: Mesenchymal stem cells seeded onto tissue-engineered osteoinductive scaffolds enhance the healing process of critical-sized radial bone defects in rat. Cell Tissue Res 2018; 374:63-81. [DOI: 10.1007/s00441-018-2837-7] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2017] [Accepted: 03/28/2018] [Indexed: 01/20/2023]
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Bougioukli S, Sugiyama O, Pannell W, Ortega B, Tan MH, Tang AH, Yoho R, Oakes DA, Lieberman JR. Gene Therapy for Bone Repair Using Human Cells: Superior Osteogenic Potential of Bone Morphogenetic Protein 2-Transduced Mesenchymal Stem Cells Derived from Adipose Tissue Compared to Bone Marrow. Hum Gene Ther 2018; 29:507-519. [PMID: 29212377 DOI: 10.1089/hum.2017.097] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Ex vivo regional gene therapy strategies using animal mesenchymal stem cells genetically modified to overexpress osteoinductive growth factors have been successfully used in a variety of animal models to induce both heterotopic and orthotopic bone formation. However, in order to adapt regional gene therapy for clinical applications, it is essential to assess the osteogenic capacity of transduced human cells and choose the cell type that demonstrates the best clinical potential. Bone-marrow stem cells (BMSC) and adipose-derived stem cells (ASC) were selected in this study for in vitro evaluation, before and after transduction with a lentiviral two-step transcriptional amplification system (TSTA) overexpressing bone morphogenetic protein 2 (BMP-2; LV-TSTA-BMP-2) or green fluorescent protein (GFP; LV-TSTA-GFP). Cell growth, transduction efficiency, BMP-2 production, and osteogenic capacity were assessed. The study demonstrated that BMSC were characterized by a slower cell growth compared to ASC. Fluorescence-activated cell sorting analysis of GFP-transduced cells confirmed successful transduction with the vector and revealed an overall higher but not statistically significant transduction efficiency in ASC versus BMSC (90.2 ± 4.06% vs. 80.4 ± 8.51%, respectively; p = 0.146). Enzyme-linked immunosorbent assay confirmed abundant BMP-2 production by both cell types transduced with LV-TSTA-BMP-2, with BMP-2 production being significantly higher in ASC versus BMSC (239.5 ± 116.55 ng vs. 70.86 ± 24.7 ng; p = 0.001). Quantitative analysis of extracellular deposition of calcium (Alizarin red) and alkaline phosphatase activity showed that BMP-2-transduced cells had a higher osteogenic differentiation capacity compared to non-transduced cells. When comparing the two cell types, ASC/LV-TSTA-BMP-2 demonstrated a significantly higher mineralization potential compared to BMSC/LV-TSTA-BMP-2 7 days post transduction (p = 0.014). In conclusion, this study demonstrates that transduction with LV-TSTA-BMP-2 can significantly enhance the osteogenic potential of both human BMSC and ASC. BMP-2-treated ASC exhibited higher BMP-2 production and greater osteogenic differentiation capacity compared to BMP-2-treated BMSC. These results, along with the fact that liposuction is an easy procedure with lower donor-site morbidity compared to BM aspiration, indicate that adipose tissue might be a preferable source of MSCs to develop a regional gene therapy approach to treat difficult bone-repair scenarios.
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Affiliation(s)
- Sofia Bougioukli
- 1 Department of Orthopedic Surgery, Keck School of Medicine, University of Southern California , Los Angeles, California
| | - Osamu Sugiyama
- 1 Department of Orthopedic Surgery, Keck School of Medicine, University of Southern California , Los Angeles, California
| | - William Pannell
- 1 Department of Orthopedic Surgery, Keck School of Medicine, University of Southern California , Los Angeles, California
| | - Brandon Ortega
- 1 Department of Orthopedic Surgery, Keck School of Medicine, University of Southern California , Los Angeles, California
| | - Matthew H Tan
- 1 Department of Orthopedic Surgery, Keck School of Medicine, University of Southern California , Los Angeles, California
| | - Amy H Tang
- 1 Department of Orthopedic Surgery, Keck School of Medicine, University of Southern California , Los Angeles, California
| | - Robert Yoho
- 2 Cosmetic Surgery Practice , Pasadena, California
| | - Daniel A Oakes
- 1 Department of Orthopedic Surgery, Keck School of Medicine, University of Southern California , Los Angeles, California
| | - Jay R Lieberman
- 1 Department of Orthopedic Surgery, Keck School of Medicine, University of Southern California , Los Angeles, California
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Clough BH, Zeitouni S, Krause U, Chaput CD, Cross LM, Gaharwar AK, Gregory CA. Rapid Osteogenic Enhancement of Stem Cells in Human Bone Marrow Using a Glycogen-Synthease-Kinase-3-Beta Inhibitor Improves Osteogenic Efficacy In Vitro and In Vivo. Stem Cells Transl Med 2018; 7:342-353. [PMID: 29405665 PMCID: PMC5866944 DOI: 10.1002/sctm.17-0229] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2017] [Revised: 12/06/2017] [Accepted: 12/26/2017] [Indexed: 12/12/2022] Open
Abstract
Non‐union defects of bone are a major problem in orthopedics, especially for patients with a low healing capacity. Fixation devices and osteoconductive materials are used to provide a stable environment for osteogenesis and an osteogenic component such as autologous human bone marrow (hBM) is then used, but robust bone formation is contingent on the healing capacity of the patients. A safe and rapid procedure for improvement of the osteoanabolic properties of hBM is, therefore, sought after in the field of orthopedics, especially if it can be performed within the temporal limitations of the surgical procedure, with minimal manipulation, and at point‐of‐care. One way to achieve this goal is to stimulate canonical Wingless (cWnt) signaling in bone marrow‐resident human mesenchymal stem cells (hMSCs), the presumptive precursors of osteoblasts in bone marrow. Herein, we report that the effects of cWnt stimulation can be achieved by transient (1–2 hours) exposure of osteoprogenitors to the GSK3β‐inhibitor (2′Z,3′E)‐6‐bromoindirubin‐3′‐oxime (BIO) at a concentration of 800 nM. Very‐rapid‐exposure‐to‐BIO (VRE‐BIO) on either hMSCs or whole hBM resulted in the long‐term establishment of an osteogenic phenotype associated with accelerated alkaline phosphatase activity and enhanced transcription of the master regulator of osteogenesis, Runx2. When VRE‐BIO treated hBM was tested in a rat spinal fusion model, VRE‐BIO caused the formation of a denser, stiffer, fusion mass as compared with vehicle treated hBM. Collectively, these data indicate that the VRE‐BIO procedure may represent a rapid, safe, and point‐of‐care strategy for the osteogenic enhancement of autologous hBM for use in clinical orthopedic procedures. stemcellstranslationalmedicine2018;7:342–353
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Affiliation(s)
- Bret H Clough
- Department of Molecular and Cellular Medicine, Institute for Regenerative Medicine, Texas A&M Health Science Center, College Station, Texas, USA
| | - Suzanne Zeitouni
- Department of Molecular and Cellular Medicine, Institute for Regenerative Medicine, Texas A&M Health Science Center, College Station, Texas, USA
| | - Ulf Krause
- Institute for Transfusion Medicine and Transplant Immunology, University Hospital Muenster, Muenster, Germany
| | - Christopher D Chaput
- Department of Orthopedic Surgery, Baylor Scott and White Hospital, Temple, Texas, USA
| | - Lauren M Cross
- Department of Biomedical Engineering, Texas A&M University, College Station, Texas, USA
| | - Akhilesh K Gaharwar
- Department of Biomedical Engineering, Texas A&M University, College Station, Texas, USA.,Department of Material Sciences, College Station, Texas, USA.,Center for Remote Health Technologies and Systems, Texas A&M University, College Station, Texas, USA
| | - Carl A Gregory
- Department of Molecular and Cellular Medicine, Institute for Regenerative Medicine, Texas A&M Health Science Center, College Station, Texas, USA
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Huang YZ, Cai JQ, Xue J, Chen XH, Zhang CL, Li XQ, Yang ZM, Huang YC, Deng L. The Poor Osteoinductive Capability of Human Acellular Bone Matrix. Int J Artif Organs 2018. [DOI: 10.1177/039139881203501204] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
Demineralized bone matrix (DBM) has extensive clinical use for bone regeneration because of its osteoinductive and osteoconductive aptitude. It is suggested that the demineralization process in bone matrix preparation is influential in maintaining osteoinductivity; however, relevant investigations, especially into the osteoinductivity of acellular bone matrix, are not often performed. This study addressed the osteoinductive capability of human acellular cancellous bone matrix (ACBM) after subcutaneous implantation in a rat model. The growth and osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (rBM-MSCs) seeded in this material were also studied. Without the demineralization process, the ACBM we obtained had an interconnected porous network and the micropores in the surface were clearly exposed. After the ACBM was subcutaneously implanted for 4 months, new osteoid formation was noted but not typical mature bone formation. rBM-MSCs grew well in the ACBM and kept a steady morphology after continuous culture for 28 days. However, no mineralized nodule formation was detected and the expression levels of genes encoding osteogenic markers were significantly decreased. These results demonstrated that human ACBM possess the structural features of native bone and poor osteoinductivity; nonetheless this material helped to preserve the undifferentiated phenotype of rBM-MSCs. Such insights may further broaden our understanding of the application of ACBM for bone regeneration and the creation of stem cell niches.
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Affiliation(s)
- Yi-Zhou Huang
- Laboratory of Stem Cell and Tissue Engineering, State Key Laboratory of Biotherapy and Regenerative Medicine Research Center, West China Hospital, Sichuan University, Chengdu - P.R. China
| | - Jia-Qin Cai
- Laboratory of Stem Cell and Tissue Engineering, State Key Laboratory of Biotherapy and Regenerative Medicine Research Center, West China Hospital, Sichuan University, Chengdu - P.R. China
| | - Jing Xue
- State Key Laboratory of Oral Diseases, Sichuan University, Chengdu - P.R. China
| | - Xiao-He Chen
- Laboratory of Stem Cell and Tissue Engineering, State Key Laboratory of Biotherapy and Regenerative Medicine Research Center, West China Hospital, Sichuan University, Chengdu - P.R. China
| | - Chao-Liang Zhang
- State Key Laboratory of Oral Diseases, Sichuan University, Chengdu - P.R. China
| | - Xiu-Qun Li
- Laboratory of Stem Cell and Tissue Engineering, State Key Laboratory of Biotherapy and Regenerative Medicine Research Center, West China Hospital, Sichuan University, Chengdu - P.R. China
| | - Zhi-Ming Yang
- Laboratory of Stem Cell and Tissue Engineering, State Key Laboratory of Biotherapy and Regenerative Medicine Research Center, West China Hospital, Sichuan University, Chengdu - P.R. China
| | - Yong-Can Huang
- Laboratory of Stem Cell and Tissue Engineering, State Key Laboratory of Biotherapy and Regenerative Medicine Research Center, West China Hospital, Sichuan University, Chengdu - P.R. China
- Department of Orthopaedics and Traumatology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong - P.R. China
| | - Li Deng
- Laboratory of Stem Cell and Tissue Engineering, State Key Laboratory of Biotherapy and Regenerative Medicine Research Center, West China Hospital, Sichuan University, Chengdu - P.R. China
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Cao H, Sun ZB, Zhang L, Qian W, Li CY, Guo XP, Zhang Y. Adenovirus-mediated bone morphogenetic protein-2 promotes osteogenic differentiation in human mesenchymal stem cells in vitro. Exp Ther Med 2017; 14:377-382. [PMID: 28672942 DOI: 10.3892/etm.2017.4482] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2015] [Accepted: 03/17/2017] [Indexed: 01/18/2023] Open
Abstract
Delayed and failed bone union following fracture is a common clinical complication that requires treatment in orthopedics. Cell-based therapies and tissue-engineering approaches are potential therapeutic strategies for bone repair and fracture healing. However, the effect of adenovirus expressing bone morphogenetic protein-2 (Ad-BMP-2) on the osteogenic ability of human mesenchymal stem cells (hMSCs) has remained to be fully elucidated. Therefore, in the present study, hMSCs were transduced using Ad-BMP-2 to assess the effects of its application and to determine whether Ad-BMP-2 promotes the osteogenic differentiation of hMSCs. The purity of the hMSC cultures was assessed using flow cytometric analysis. In order to assess the osteogenic activity, alkaline phosphatase activity (ALP) was measured and to estimate the osteoblastic mineralization and calcification, von Kossa staining for phosphates was performed. Cells positive for Src homology 2 domain were determined to be hMSCs and the presence of CD34 was used to distinguish hematopoietic lineages. Following treatment, the Ad-BMP-2 and control group had significantly increased ALP levels (P<0.05). Compared to the blank group and the group transfected with adenoviral vector containing LacZ, the phosphate deposition in the Ad-BMP-2 group and the positive control group treated with dexamethasone was markedly increased. The results of the present study suggested that Ad-BMP-2 promotes osteogenic differentiation in hMSCs and may have a potential application in treating delayed union and nonunion following bone fracture.
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Affiliation(s)
- Hong Cao
- Department of Orthopedic Surgery, Renmin Hospital, Hubei University of Medicine, Shiyan, Hubei 442000, P.R. China
| | - Zhi-Bo Sun
- Department of Orthopedic Surgery, Renmin Hospital, Hubei University of Medicine, Shiyan, Hubei 442000, P.R. China
| | - Lei Zhang
- Department of Orthopedic Surgery, Renmin Hospital, Hubei University of Medicine, Shiyan, Hubei 442000, P.R. China
| | - Wei Qian
- Department of Orthopedic Surgery, Renmin Hospital, Hubei University of Medicine, Shiyan, Hubei 442000, P.R. China
| | - Chun-Yang Li
- Department of Reproductive Medicine, Renmin Hospital, Hubei University of Medicine, Shiyan, Hubei 442000, P.R. China
| | - Xiao-Peng Guo
- Department of Orthopedic Surgery, Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei 442000, P.R. China
| | - Ying Zhang
- Department of Reproductive Medicine, Renmin Hospital, Hubei University of Medicine, Shiyan, Hubei 442000, P.R. China
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The Holy Grail of Orthopedic Surgery: Mesenchymal Stem Cells-Their Current Uses and Potential Applications. Stem Cells Int 2017; 2017:2638305. [PMID: 28698718 PMCID: PMC5494105 DOI: 10.1155/2017/2638305] [Citation(s) in RCA: 73] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2017] [Accepted: 04/16/2017] [Indexed: 02/07/2023] Open
Abstract
Only select tissues and organs are able to spontaneously regenerate after disease or trauma, and this regenerative capacity diminishes over time. Human stem cell research explores therapeutic regenerative approaches to treat various conditions. Mesenchymal stem cells (MSCs) are derived from adult stem cells; they are multipotent and exert anti-inflammatory and immunomodulatory effects. They can differentiate into multiple cell types of the mesenchyme, for example, endothelial cells, osteoblasts, chondrocytes, fibroblasts, tenocytes, vascular smooth muscle cells, and sarcomere muscular cells. MSCs are easily obtained and can be cultivated and expanded in vitro; thus, they represent a promising and encouraging treatment approach in orthopedic surgery. Here, we review the application of MSCs to various orthopedic conditions, namely, orthopedic trauma; muscle injury; articular cartilage defects and osteoarthritis; meniscal injuries; bone disease; nerve, tendon, and ligament injuries; spinal cord injuries; intervertebral disc problems; pediatrics; and rotator cuff repair. The use of MSCs in orthopedics may transition the practice in the field from predominately surgical replacement and reconstruction to bioregeneration and prevention. However, additional research is necessary to explore the safety and effectiveness of MSC treatment in orthopedics, as well as applications in other medical specialties.
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Clough BH, McNeill EP, Palmer D, Krause U, Bartosh TJ, Chaput CD, Gregory CA. An allograft generated from adult stem cells and their secreted products efficiently fuses vertebrae in immunocompromised athymic rats and inhibits local immune responses. Spine J 2017; 17:418-430. [PMID: 27765715 PMCID: PMC5309156 DOI: 10.1016/j.spinee.2016.10.009] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/23/2015] [Revised: 09/21/2016] [Accepted: 10/12/2016] [Indexed: 02/06/2023]
Abstract
BACKGROUND CONTEXT Spine pain and the disability associated with it are epidemic in the United States. According to the National Center for Health Statistics, more than 650,000 spinal fusion surgeries are performed annually in the United States, and yet there is a failure rate of 15%-40% when standard methods employing current commercial bone substitutes are used. Autologous bone graft is the gold standard in terms of fusion success, but the morbidity associated with the procedure and the limitations in the availability of sufficient material have limited its use in the majority of cases. A freely available and immunologically compatible bone mimetic with the properties of live tissue is likely to substantially improve the outcome of spine fusion procedures without the disadvantages of autologous bone graft. PURPOSE This study aimed to compare a live human bone tissue analog with autologous bone grafting in an immunocompromised rat model of posterolateral fusion. DESIGN/SETTING This is an in vitro and in vivo preclinical study of a novel human stem cell-derived construct for efficacy in posterolateral lumbar spine fusion. METHODS Osteogenically enhanced human mesenchymal stem cells (OEhMSCs) were generated by exposure to conditions that activate the early stages of osteogenesis. Immunologic characteristics of OEhMSCs were evaluated in vitro. The secreted extracellular matrix from OEhMSCs was deposited on a clinical-grade gelatin sponge, resulting in bioconditioned gelatin sponge (BGS). Bioconditioned gelatin sponge was used alone, with live OEhMSCs (BGS+OEhMSCs), or with whole human bone marrow (BGS+hBM). Efficacy for spine fusion was determined by an institutionally approved animal model using 53 nude rats. RESULTS Bioconditioned gelatin sponge with live OEhMSCs did not cause cytotoxicity when incubated with immunologically mismatched lymphocytes, and OEhMSCs inhibited lymphocyte expansion in mixed lymphocyte assays. Bioconditioned gelatin sponge with live OEhMSC and BGS+hBM constructs induced profound bone growth at fusion sites in vivo, with a comparable rate of fusion with syngeneic bone graft (negative [0 of 10], BGS alone [0 of 10], bone graft [7 of 10], BGS+OEhMSC [10 of 15], and BGS+hBM [8 of 8]). CONCLUSIONS Collectively, these studies demonstrate that BGS+OEhMSC constructs possess low immunogenicity and drive vertebral fusion with efficiency matching syngeneic bone graft in rodents. We also demonstrate that BGS serves as a promising scaffold for spine fusion when combined with hBM.
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Affiliation(s)
- Bret H. Clough
- Institute for Regenerative Medicine, Texas A&M Health Science Center, 206 Olsen Blvd, Room 228 MS1114, College Station, TX 77845, USA
| | - Eoin P. McNeill
- Institute for Regenerative Medicine, Texas A&M Health Science Center, 206 Olsen Blvd, Room 228 MS1114, College Station, TX 77845, USA
| | - Daniel Palmer
- Institute for Regenerative Medicine, Texas A&M Health Science Center, 206 Olsen Blvd, Room 228 MS1114, College Station, TX 77845, USA
| | - Ulf Krause
- Department of Orthopedic Surgery, Baylor Scott and White Hospital, Texas A&M Health Science Center, 2401 S. 31st St, Temple, TX 76508, USA,Institute for Transfusion Medicine and Transplant Immunology, University Hospital Muenster, 11 Domagkstr, Muenster 48149, Germany
| | - Thomas J. Bartosh
- Institute for Regenerative Medicine, Texas A&M Health Science Center, 206 Olsen Blvd, Room 228 MS1114, College Station, TX 77845, USA
| | - Christopher D. Chaput
- Department of Orthopedic Surgery, Baylor Scott and White Hospital, Texas A&M Health Science Center, 2401 S. 31st St, Temple, TX 76508, USA
| | - Carl A. Gregory
- Institute for Regenerative Medicine, Texas A&M Health Science Center, 206 Olsen Blvd, Room 228 MS1114, College Station, TX 77845, USA,Corresponding author. Institute for Regenerative Medicine, Texas A&M Health Science Center, 206 Olsen Blvd, Room 228 MS1114, College Station, TX 77845, USA. Tel.: (979) 436-9643; fax: (979) 436-9679. (C.A. Gregory)
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Pytlík R, Rentsch C, Soukup T, Novotný L, Rentsch B, Kanderová V, Rychtrmocová H, Kalmárová M, Stehlík D, Trněný M, Slanař O. Efficacy and safety of human mesenchymal stromal cells in healing of critical-size bone defects in immunodeficient rats. Physiol Res 2016; 66:113-123. [PMID: 27782744 DOI: 10.33549/physiolres.933376] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022] Open
Abstract
To evaluate the preclinical efficacy and safety of human mesenchymal stem cells (hMSC) rapidly expanded in growth medium for clinical use with human serum and recombinant growth factors, we conducted a controlled, randomized trial of plasma clots with hMSC vs. plasma clots only in critical segmental femoral defects in rnu/rnu immunodeficient rats. X-ray, microCT and histomorphometrical evaluation were performed at 8 and 16 weeks. MSC were obtained from healthy volunteers and patients with lymphoid malignancy. Human MSC survived in the defect for the entire duration of the trial. MSC from healthy volunteers, in contrast to hMSC from cancer patients, significantly improved bone healing at 8, but not 16 weeks. However, at 16 weeks, hMSC significantly improved vasculogenesis in residual defect. We conclude that hMSC from healthy donors significantly contributed to the healing of bone defects at 8 weeks and to the vascularisation of residual connective tissue for up to 16 weeks. We found the administration of hMSC to be safe, as no adverse reaction to human cells at the site of implantation and no evidence of migration of hMSC to distant organs was detected.
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Affiliation(s)
- R Pytlík
- First Department of Medicine, General University Hospital and First Medical Faculty of Charles University, Prague, Czech Republic.
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Ritz U, Götz H, Baranowski A, Heid F, Rommens PM, Hofmann A. Influence of different calcium phosphate ceramics on growth and differentiation of cells in osteoblast-endothelial co-cultures. J Biomed Mater Res B Appl Biomater 2016; 105:1950-1962. [PMID: 27292649 DOI: 10.1002/jbm.b.33728] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2016] [Revised: 05/02/2016] [Accepted: 05/24/2016] [Indexed: 12/19/2022]
Abstract
Strategies for improvement of angiogenesis and vasculogenesis using different cells and materials are paramount aims in the field of bone tissue engineering. Thereby, the interaction between different cell types and scaffold materials is crucial for growth, differentiation, and long-term outcomes of tissue-engineered constructs. In this study, we evaluated the interaction of osteoblasts and endothelial cells in three-dimensional tissue-engineered constructs using beta tricalciumphosphate (β-TCP, [ß-Ca3 (PO4 )2 ]) and calcium-deficient hydroxyapatite (CDHA, [Ca9 (PO4 )5 (HPO4 )OH]) ceramics as scaffolds. We focused on initial cell organization, cell proliferation, and differential expression of osteoblastic and endothelial markers employing monocultures and co-cultures of endothelial cells of two different origins [human umbilical vein endothelial cells (HUVECs) and outgrowth endothelial cells (OECs)] with primary human osteoblasts (hOBs). Despite different chemical and physical characteristics of CDHA and β-TCP ceramics, similar patterns in cell growth, differentiation, and gene expression were detected in tissue-engineered constructs consisting of hOB, HUVEC, and HUVEC/hOB-co-cultures. Under dynamic cell culture conditions we found proliferation of these cells with stable endothelial and osteoblastic differentiation patterns. Both material types are highly biocompatible with these cells providing a promising perspective for the future research. In this study, both materials did not support growth and differentiation of OEC. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1950-1962, 2017.
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Affiliation(s)
- Ulrike Ritz
- Department of Orthopedics and Traumatology, University Medical Centre, Johannes Gutenberg University Mainz, Mainz, Germany
| | - Hermann Götz
- Platform for Biomaterial Research, University Medical Centre, Johannes Gutenberg University Mainz, Mainz, Germany
| | - Andreas Baranowski
- Department of Orthopedics and Traumatology, University Medical Centre, Johannes Gutenberg University Mainz, Mainz, Germany
| | - Florian Heid
- Department of Anesthesiology, University Medical Centre, Johannes Gutenberg University Mainz, Mainz, Germany
| | - Pol Maria Rommens
- Department of Orthopedics and Traumatology, University Medical Centre, Johannes Gutenberg University Mainz, Mainz, Germany
| | - Alexander Hofmann
- Department of Orthopedics and Traumatology, University Medical Centre, Johannes Gutenberg University Mainz, Mainz, Germany
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Houdek MT, Wyles CC, Packard BD, Terzic A, Behfar A, Sierra RJ. Decreased Osteogenic Activity of Mesenchymal Stem Cells in Patients With Corticosteroid-Induced Osteonecrosis of the Femoral Head. J Arthroplasty 2016; 31:893-8. [PMID: 26404846 DOI: 10.1016/j.arth.2015.08.017] [Citation(s) in RCA: 86] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/14/2015] [Revised: 08/18/2015] [Accepted: 08/26/2015] [Indexed: 02/01/2023] Open
Abstract
BACKGROUND Osteonecrosis (ON) of the femoral head occurs when cells of trabecular bone spontaneously die. Mesenchymal stem cells (MSCs) have been introduced into the femoral head in an attempt to halt progression of the disease. The purpose of this study was to functionally compare MSCs in patients with ON of the femoral head with patients without. METHODS Mesenchymal stem cells were isolated from 20 patients with corticosteroid-induced ON and 10 controls without. Colony-forming unit and proliferation assays were used to assess MSC proliferation. Mesenchymal stem cells were differentiated into bone, fat, and cartilage. Functional assays were used to quantify the differentiation capacity. RESULTS Control MSCs demonstrated greater cellular growth potential and improved ability to differentiate into bone. CONCLUSION The decreased ability to differentiate into bone may be a reason why patients treated with autologous MSC infusion fail regenerative treatment strategies and progress to collapse.
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Affiliation(s)
- Matthew T Houdek
- Department of Orthopedic Surgery, Mayo Clinic, Rochester, Minnesota; Center for Regenerative Medicine, Mayo Clinic, Rochester, Minnesota
| | - Cody C Wyles
- Mayo Medical School, Mayo Clinic, Rochester, Minnesota; Center for Regenerative Medicine, Mayo Clinic, Rochester, Minnesota
| | | | - Andre Terzic
- Division of Cardiovascular Diseases, Mayo Clinic, Rochester, Minnesota; Center for Regenerative Medicine, Mayo Clinic, Rochester, Minnesota
| | - Atta Behfar
- Division of Cardiovascular Diseases, Mayo Clinic, Rochester, Minnesota; Center for Regenerative Medicine, Mayo Clinic, Rochester, Minnesota
| | - Rafael J Sierra
- Department of Orthopedic Surgery, Mayo Clinic, Rochester, Minnesota; Center for Regenerative Medicine, Mayo Clinic, Rochester, Minnesota
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Tillotson M, Logan N, Brett P. Osteogenic stem cell selection for repair and regeneration. Bone Rep 2016; 5:22-32. [PMID: 28326344 PMCID: PMC4926815 DOI: 10.1016/j.bonr.2016.01.003] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/24/2015] [Revised: 12/23/2015] [Accepted: 01/24/2016] [Indexed: 12/20/2022] Open
Abstract
The first osteogenic cells to attach to a titanium (Ti) implant after placement are the multipotent stromal cells (MSCs) that circulate in the bloodstream and are recruited to the site of tissue damage. The reservoirs of these cells are heterogeneous in nature, consisting of a mixture of cells with varying differentiation abilities. In order to utilise these cells and to reduce the chance of unwanted events during regenerative therapies, the selection of a subset of cells that is truly multipotent is required. The behaviour of these cells has been shown to be altered by modifications to Ti implant surfaces, most notably rough, hydrophilic Ti. These changes in behaviour underpin the differences seen in clinical performance of these surfaces. In this study Human bone marrow derived stromal cells (hBMSCs) have been cultured on modified Ti surfaces in order to analyse these changes in cell behaviour. The results demonstrate the different effects of the surfaces and suggest that one surface selectively enriches the population with osteogenic adult ‘stem cells’ by inducing the cell death of the more differentiated cells. Combined with subsequent expansion in bioreactors before implantation, this may lead to a new source of cells for regenerative therapies.
Different titanium surfaces elicit differing responses from bone marrow derived stromal cells. Hydrophilic rough titanium induces increased apoptosis and necrosis in MSCs in vitro. Cells selected on rough hydrophilic titanium are more osteogenic than the parent population. This may lead to a new source of osteogenic cells for regenerative therapies.
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Affiliation(s)
- Marcus Tillotson
- Biomaterials and Tissue Engineering, UCL Eastman Dental Institute, 256 Gray's Inn Road, London WC1X 8LD, United Kingdom
| | - Niall Logan
- Biomaterials and Tissue Engineering, UCL Eastman Dental Institute, 256 Gray's Inn Road, London WC1X 8LD, United Kingdom
| | - Peter Brett
- Biomaterials and Tissue Engineering, UCL Eastman Dental Institute, 256 Gray's Inn Road, London WC1X 8LD, United Kingdom
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Gianakos A, Ni A, Zambrana L, Kennedy JG, Lane JM. Bone Marrow Aspirate Concentrate in Animal Long Bone Healing: An Analysis of Basic Science Evidence. J Orthop Trauma 2016; 30:1-9. [PMID: 26371620 DOI: 10.1097/bot.0000000000000453] [Citation(s) in RCA: 49] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
OBJECTIVES Long bone fractures that fail to heal or show a delay in healing can lead to increased morbidity. Bone marrow aspirate concentrate (BMAC) containing bone mesenchymal stem cells (BMSCs) has been suggested as an autologous biologic adjunct to aid long bone healing. The purpose of this study was to systematically review the basic science in vivo evidence for the use of BMAC with BMSCs in the treatment of segmental defects in animal long bones. DATA SOURCES The PubMed/MEDLINE and EMBASE databases were screened in July 14-25, 2014. STUDY SELECTION The following search criteria were used: [("bmac" OR "bone marrow aspirate concentrate" OR "bmc" OR "bone marrow concentrate" OR "mesenchymal stem cells") AND ("bone" OR "osteogenesis" OR "fracture healing" OR "nonunion" OR "delayed union")]. DATA EXTRACTION Three authors extracted data and analyzed for trends. Quality of evidence score was given to each study. DATA SYNTHESIS Results are presented as Hedge G standardized effect sizes with 95% confidence intervals. RESULTS The search yielded 35 articles for inclusion. Of studies reporting statistics, 100% showed significant increase in bone formation in the BMAC group on radiograph. Ninety percent reported significant improvement in earlier bone healing on histologic/histomorphometric assessment. Eighty-one percent reported a significant increase in bone area on micro-computed tomography. Seventy-eight percent showed a higher torsional stiffness for the BMAC-treated defects. CONCLUSION In the in vivo studies evaluated, BMAC confer beneficial effects on the healing of segmental defects in animal long bone models when compared with a control. Proof-of-concept has been established for BMAC in the treatment of animal segmental bone defects.
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Abstract
Stem cells offer great promise to help understand the normal mechanisms of tissue renewal, regeneration, and repair, and also for development of cell-based therapies to treat patients after tissue injury. Most adult tissues contain stem cells and progenitor cells that contribute to homeostasis, remodeling, and repair. Multiple stem and progenitor cell populations in bone are found in the marrow, the endosteum, and the periosteum. They contribute to the fracture healing process after injury and are an important component in tissue engineering approaches for bone repair. This review focuses on current concepts in stem cell biology related to fracture healing and bone tissue regeneration, as well as current strategies and limitations for clinical cell-based therapies.
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Tinsley BA, Dukas A, Pensak MJ, Adams DJ, Tang AH, Ominsky MS, Ke HZ, Lieberman JR. Systemic Administration of Sclerostin Antibody Enhances Bone Morphogenetic Protein-Induced Femoral Defect Repair in a Rat Model. J Bone Joint Surg Am 2015; 97:1852-9. [PMID: 26582615 DOI: 10.2106/jbjs.o.00171] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
BACKGROUND Recombinant human bone morphogenetic protein (rhBMP)-2 is a potent osteoinductive agent; however, its clinical use has been reduced because of safety and efficacy concerns. In preclinical studies involving a critical-sized defect in a rat model, sclerostin antibody (Scl-Ab) treatment increased bone formation within the defect but did not result in reliable healing. The purpose of the current study was to evaluate bone repair of a critical-sized femoral defect in a rat model with use of local implantation of rhBMP-2 combined with systemic administration of Scl-Ab. METHODS A critical-sized femoral defect was created in rats randomized into three treatment groups: local rhBMP-2 and systemic Scl-Ab (Scl + BMP), local rhBMP-2 alone, and collagen sponge alone (operative control). The Scl + BMP group received local rhBMP-2 (10 μg) on a collagen sponge placed within the defect intraoperatively and then twice weekly injections of Scl-Ab (25 mg/kg) administered postoperatively. The femora were evaluated at twelve weeks with use of radiography, microcomputed tomography (microCT), histomorphometric analysis, and biomechanical testing. RESULTS At twelve weeks, all Scl + BMP and rhBMP-2 only samples were healed. No femora healed in the operative control group. Histomorphometric analysis demonstrated more bone in the Scl + BMP samples than in the samples treated with rhBMP-2 alone (p = 0.029) and the control samples (p = 0.003). MicroCT revealed that the Scl + BMP group had a 90% greater bone volume within the defect region compared with the rhBMP-2 group and a 350% greater bone volume compared with the operative control group (p < 0.001). Biomechanical testing showed that the group treated with Scl + BMP had greater torsional strength and rigidity compared with the rhBMP-2 group (p < 0.001 and p = 0.047) and the intact femoral control group (p < 0.001). Torque to failure was lower in the rhBMP-2 group compared with the intact femoral control group (p < 0.002). Mean energy to failure was higher in the Scl + BMP samples compared with the rhBMP-2 only samples (p = 0.001). CONCLUSIONS In a critical-sized femoral defect in a rat model, local rhBMP-2 combined with systemic administration of Scl-Ab resulted in more robust healing that was stronger and more rigid than results for rhBMP-2 alone and intact nonoperative femora. CLINICAL RELEVANCE Our study demonstrated that combining an osteoinductive agent with a systemically administered antibody that promotes bone formation can enhance bone repair and has potential as a therapeutic regimen in humans.
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Affiliation(s)
- Brian A Tinsley
- Department of Orthopaedic Surgery, University of Connecticut Health Center, MARB 4th floor, 263 Farmington Avenue, Farmington, CT 06030. E-mail address for B.A. Tinsley:
| | - Alex Dukas
- Department of Orthopaedic Surgery, University of Connecticut Health Center, MARB 4th floor, 263 Farmington Avenue, Farmington, CT 06030. E-mail address for B.A. Tinsley:
| | - Michael J Pensak
- Department of Orthopaedic Surgery, University of Connecticut Health Center, MARB 4th floor, 263 Farmington Avenue, Farmington, CT 06030. E-mail address for B.A. Tinsley:
| | - Douglas J Adams
- Department of Orthopaedic Surgery, University of Connecticut Health Center, MARB 4th floor, 263 Farmington Avenue, Farmington, CT 06030. E-mail address for B.A. Tinsley:
| | - Amy H Tang
- Department of Orthopaedic Surgery, Keck School of Medicine, University of Southern California, 1520 San Pablo Street, Los Angeles, CA 90033
| | - Michael S Ominsky
- Department of Metabolic Disorders, Amgen, Inc., 1 Amgen Center Drive, Thousand Oaks, CA 91320
| | - Hua Zhu Ke
- Bone Research, UCB Pharma, 208 Bath Road, Slough, Berkshire, SL1 3WE, United Kingdom
| | - Jay R Lieberman
- Department of Orthopaedic Surgery, Keck School of Medicine, University of Southern California, 1520 San Pablo Street, Los Angeles, CA 90033
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Lee S, Zhang X, Shen J, James AW, Chung CG, Hardy R, Li C, Girgius C, Zhang Y, Stoker D, Wang H, Wu BM, Peault B, Ting K, Soo C. Brief Report: Human Perivascular Stem Cells and Nel-Like Protein-1 Synergistically Enhance Spinal Fusion in Osteoporotic Rats. Stem Cells 2015; 33:3158-63. [PMID: 26173400 DOI: 10.1002/stem.2103] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2015] [Revised: 05/26/2015] [Accepted: 06/01/2015] [Indexed: 01/09/2023]
Abstract
Autologous bone grafts (ABGs) are considered as the gold standard for spinal fusion. However, osteoporotic patients are poor candidates for ABGs due to limited osteogenic stem cell numbers and function of the bone microenvironment. There is a need for stem cell-based spinal fusion of proven efficacy under either osteoporotic or nonosteoporotic conditions. The purpose of this study is to determine the efficacy of human perivascular stem cells (hPSCs), a population of mesenchymal stem cells isolated from adipose tissue, in the presence and absence of NELL-1, an osteogenic protein, for spinal fusion in the osteoporosis. Osteogenic differentiation of hPSCs with and without NELL-1 was tested in vitro. The results indicated that NELL-1 significantly increased the osteogenic potential of hPSCs in both osteoporotic and nonosteoporotic donors. Next, spinal fusion was performed by implanting scaffolds with regular or high doses of hPSCs, with or without NELL-1 in ovariectomized rats (n = 41). Regular doses of hPSCs or NELL-1 achieved the fusion rates of only 20%-37.5% by manual palpation. These regular doses had previously been shown to be effective in nonosteoporotic rat spinal fusion. Remarkably, the high dose of hPSCs+NELL-1 significantly improved the fusion rates among osteoporotic rats up to approximately 83.3%. Microcomputed tomography imaging and quantification further confirmed solid bony fusion with high dose hPSCs+NELL-1. Finally, histologically, direct in situ involvement of hPSCs in ossification was shown using undecalcified samples. To conclude, hPSCs combined with NELL-1 synergistically enhances spinal fusion in osteoporotic rats and has great potential as a novel therapeutic strategy for osteoporotic patients.
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Affiliation(s)
- Soonchul Lee
- Department of Orthopaedic Surgery, CHA Bundang Medical Center, CHA University School of Medicine, Gyeonggi-do, Republic of Korea.,UCLA and Orthopaedic Hospital Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, University of California, Los Angeles, California, USA
| | - Xinli Zhang
- Division of Growth and Development, School of Dentistry, University of California, Los Angeles, California, USA
| | - Jia Shen
- Division of Growth and Development, School of Dentistry, University of California, Los Angeles, California, USA
| | - Aaron W James
- Department of Pathology and Laboratory Medicine, University of California, Los Angeles, California, USA
| | - Choon G Chung
- Division of Growth and Development, School of Dentistry, University of California, Los Angeles, California, USA
| | - Reef Hardy
- UCLA and Orthopaedic Hospital Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, University of California, Los Angeles, California, USA
| | - Chenshuang Li
- Division of Growth and Development, School of Dentistry, University of California, Los Angeles, California, USA
| | - Caroline Girgius
- Division of Growth and Development, School of Dentistry, University of California, Los Angeles, California, USA
| | - Yulong Zhang
- Department of Bioengineering, University of California, Los Angeles, California, USA
| | - David Stoker
- Marina Plastic Surgery Associates, Marina del Rey, California, USA
| | - Huiming Wang
- Affiliated Hospital of Stomatology, Medical College, Zhejiang University, Hangzhou, People's Republic of China
| | - Benjamin M Wu
- UCLA and Orthopaedic Hospital Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, University of California, Los Angeles, California, USA.,Department of Bioengineering, University of California, Los Angeles, California, USA.,Department of Materials Science and Engineering, and Division of Advanced Prosthodontics, University of California, Los Angeles, California, USA
| | - Bruno Peault
- UCLA and Orthopaedic Hospital Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, University of California, Los Angeles, California, USA.,Center For Cardiovascular Science and MRC Center for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom
| | - Kang Ting
- UCLA and Orthopaedic Hospital Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, University of California, Los Angeles, California, USA.,Division of Growth and Development, School of Dentistry, University of California, Los Angeles, California, USA
| | - Chia Soo
- UCLA and Orthopaedic Hospital Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, University of California, Los Angeles, California, USA.,UCLA Division of Plastic Surgery and Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, University of California, Los Angeles, California, USA
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Clough BH, McCarley MR, Krause U, Zeitouni S, Froese JJ, McNeill EP, Chaput CD, Sampson HW, Gregory CA. Bone regeneration with osteogenically enhanced mesenchymal stem cells and their extracellular matrix proteins. J Bone Miner Res 2015; 30:83-94. [PMID: 25130615 PMCID: PMC4280327 DOI: 10.1002/jbmr.2320] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/16/2014] [Revised: 07/02/2014] [Accepted: 07/16/2014] [Indexed: 11/09/2022]
Abstract
Although bone has remarkable regenerative capacity, about 10% of long bone fractures and 25% to 40% of vertebral fusion procedures fail to heal. In such instances, a scaffold is employed to bridge the lesion and accommodate osteoprogenitors. Although synthetic bone scaffolds mimic some of the characteristics of bone matrix, their effectiveness can vary because of biological incompatibility. Herein, we demonstrate that a composite prepared with osteogenically enhanced mesenchymal stem cells (OEhMSCs) and their extracellular matrix (ECM) has an unprecedented capacity for the repair of critical-sized defects of murine femora. Furthermore, OEhMSCs do not cause lymphocyte activation, and ECM/OEhMSC composites retain their in vivo efficacy after cryopreservation. Finally, we show that attachment to the ECM by OEhMSCs stimulates the production of osteogenic and angiogenic factors. These data demonstrate that composites of OEhMSCs and their ECM could be utilized in the place of autologous bone graft for complex orthopedic reconstructions.
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Affiliation(s)
- Bret H. Clough
- Institute for Regenerative Medicine at Scott and White Hospital, Texas A&M Health Science Center, Module C, 5701 Airport Road, Temple, TX 76502
| | - Matthew R. McCarley
- Department of Orthopedic Surgery, Scott and White Hospital, Texas A&M Health Science Center, 2401 S. 31st Street, Temple, TX 76508
- University of Texas Medical Branch, Department of Orthopedic Surgery, 301 University Blvd. Galveston, TX 77555
| | - Ulf Krause
- Department of Orthopedic Surgery, Scott and White Hospital, Texas A&M Health Science Center, 2401 S. 31st Street, Temple, TX 76508
| | - Suzanne Zeitouni
- Institute for Regenerative Medicine at Scott and White Hospital, Texas A&M Health Science Center, Module C, 5701 Airport Road, Temple, TX 76502
- University of Texas Medical Branch, Department of Orthopedic Surgery, 301 University Blvd. Galveston, TX 77555
| | - Jeremiah J. Froese
- Institute for Regenerative Medicine at Scott and White Hospital, Texas A&M Health Science Center, Module C, 5701 Airport Road, Temple, TX 76502
| | - Eoin P. McNeill
- Institute for Regenerative Medicine at Scott and White Hospital, Texas A&M Health Science Center, Module C, 5701 Airport Road, Temple, TX 76502
| | - Christopher D. Chaput
- Department of Orthopedic Surgery, Scott and White Hospital, Texas A&M Health Science Center, 2401 S. 31st Street, Temple, TX 76508
| | - H. Wayne Sampson
- Department of Medical Physiology, Texas A&M Health Science Center, 702 Southwest H.K. Dodgen Loop, Temple, TX 76504
| | - Carl A. Gregory
- Institute for Regenerative Medicine at Scott and White Hospital, Texas A&M Health Science Center, Module C, 5701 Airport Road, Temple, TX 76502
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Skovrlj B, Guzman JZ, Al Maaieh M, Cho SK, Iatridis JC, Qureshi SA. Cellular bone matrices: viable stem cell-containing bone graft substitutes. Spine J 2014; 14:2763-72. [PMID: 24929059 PMCID: PMC4402977 DOI: 10.1016/j.spinee.2014.05.024] [Citation(s) in RCA: 54] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/30/2013] [Revised: 04/03/2014] [Accepted: 05/20/2014] [Indexed: 02/03/2023]
Abstract
BACKGROUND CONTEXT Advances in the field of stem cell technology have stimulated the development and increased use of allogenic bone grafts containing live mesenchymal stem cells (MSCs), also known as cellular bone matrices (CBMs). It is estimated that CBMs comprise greater than 17% of all bone grafts and bone graft substitutes used. PURPOSE To critically evaluate CBMs, specifically their technical specifications, existing published data supporting their use, US Food and Drug Administration (FDA) regulation, cost, potential pitfalls, and other aspects pertaining to their use. STUDY DESIGN Areview of literature. METHODS A series of Ovid, Medline, and Pubmed-National Library of Medicine/National Institutes of Health (www.ncbi.nlm.nih.gov) searches were performed. Only articles in English journals or published with English language translations were included. Level of evidence of the selected articles was assessed. Specific technical information on each CBM was obtained by direct communication from the companies marketing the individual products. RESULTS Five different CBMs are currently available for use in spinal fusion surgery. There is a wide variation between the products with regard to the average donor age at harvest, total cellular concentration, percentage of MSCs, shelf life, and cell viability after defrosting. Three retrospective studies evaluating CBMs and fusion have shown fusion rates ranging from 90.2% to 92.3%, and multiple industry-sponsored trials are underway. No independent studies evaluating spinal fusion rates with the use of CBMs exist. All the commercially available CBMs claim to meet the FDA criteria under Section 361, 21 CFR Part 1271, and are not undergoing FDA premarket review. The CBMs claim to provide viable MSCs and are offered at a premium cost. Numerous challenges exist in regard to MSCs' survival, function, osteoblastic potential, and cytokine production once implanted into the intended host. CONCLUSIONS Cellular bone matrices may be a promising bone augmentation technology in spinal fusion surgery. Although CBMs appear to be safe for use as bone graft substitutes, their efficacy in spinal fusion surgery remains highly inconclusive. Large, nonindustry sponsored studies evaluating the efficacy of CBMs are required. Without results from such studies, surgeons must be made aware of the potential pitfalls of CBMs in spinal fusion surgery. With the currently available data, there is insufficient evidence to support the use of CBMs as bone graft substitutes in spinal fusion surgery.
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Affiliation(s)
- Branko Skovrlj
- Department of Neurosurgery, Icahn School of Medicine at Mount Sinai, 1 Gustave L Levy Place, Box 1136, New York, NY 10029, USA
| | - Javier Z. Guzman
- Department of Orthopaedics, Icahn School of Medicine at Mount Sinai, 5 East 98th St, 9th Floor, Box 1188, New York, NY 10029, USA
| | - Motasem Al Maaieh
- Department of Orthopaedics, Icahn School of Medicine at Mount Sinai, 5 East 98th St, 9th Floor, Box 1188, New York, NY 10029, USA
| | - Samuel K. Cho
- Department of Orthopaedics, Icahn School of Medicine at Mount Sinai, 5 East 98th St, 9th Floor, Box 1188, New York, NY 10029, USA
| | - James C. Iatridis
- Department of Orthopaedics, Icahn School of Medicine at Mount Sinai, 5 East 98th St, 9th Floor, Box 1188, New York, NY 10029, USA
| | - Sheeraz A. Qureshi
- Department of Orthopaedics, Icahn School of Medicine at Mount Sinai, 5 East 98th St, 9th Floor, Box 1188, New York, NY 10029, USA,Corresponding author. Department of Orthopaedic Surgery, Ichan School of Medicine at Mount Sinai, 5 E. 98th St, Box 1188, New York, NY 10029, USA. Tel.: (212) 241-3909; fax: (212) 534-6202.
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Hadjiargyrou M, O'Keefe RJ. The convergence of fracture repair and stem cells: interplay of genes, aging, environmental factors and disease. J Bone Miner Res 2014; 29:2307-22. [PMID: 25264148 PMCID: PMC4455538 DOI: 10.1002/jbmr.2373] [Citation(s) in RCA: 103] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/08/2014] [Revised: 08/11/2014] [Accepted: 09/10/2014] [Indexed: 01/07/2023]
Abstract
The complexity of fracture repair makes it an ideal process for studying the interplay between the molecular, cellular, tissue, and organ level events involved in tissue regeneration. Additionally, as fracture repair recapitulates many of the processes that occur during embryonic development, investigations of fracture repair provide insights regarding skeletal embryogenesis. Specifically, inflammation, signaling, gene expression, cellular proliferation and differentiation, osteogenesis, chondrogenesis, angiogenesis, and remodeling represent the complex array of interdependent biological events that occur during fracture repair. Here we review studies of bone regeneration in genetically modified mouse models, during aging, following environmental exposure, and in the setting of disease that provide insights regarding the role of multipotent cells and their regulation during fracture repair. Complementary animal models and ongoing scientific discoveries define an increasing number of molecular and cellular targets to reduce the morbidity and complications associated with fracture repair. Last, some new and exciting areas of stem cell research such as the contribution of mitochondria function, limb regeneration signaling, and microRNA (miRNA) posttranscriptional regulation are all likely to further contribute to our understanding of fracture repair as an active branch of regenerative medicine.
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Affiliation(s)
- Michael Hadjiargyrou
- Department of Life Sciences, New York Institute of Technology, Old Westbury, NY, USA
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Srijaya TC, Ramasamy TS, Kasim NHA. Advancing stem cell therapy from bench to bedside: lessons from drug therapies. J Transl Med 2014; 12:243. [PMID: 25182194 PMCID: PMC4163166 DOI: 10.1186/s12967-014-0243-9] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2014] [Accepted: 08/26/2014] [Indexed: 12/20/2022] Open
Abstract
The inadequacy of existing therapeutic tools together with the paucity of organ donors have always led medical researchers to innovate the current treatment methods or to discover new ways to cure disease. Emergence of cell-based therapies has provided a new framework through which it has given the human world a new hope. Though relatively a new concept, the pace of advancement clearly reveals the significant role that stem cells will ultimately play in the near future. However, there are numerous uncertainties that are prevailing against the present setting of clinical trials related to stem cells: like the best route of cell administration, appropriate dosage, duration and several other applications. A better knowledge of these factors can substantially improve the effectiveness of disease cure or organ repair using this latest therapeutic tool. From a certain perspective, it could be argued that by considering certain proven clinical concepts and experience from synthetic drug system, we could improve the overall efficacy of cell-based therapies. In the past, studies on synthetic drug therapies and their clinical trials have shown that all the aforementioned factors have critical ascendancy over its therapeutic outcomes. Therefore, based on the knowledge gained from synthetic drug delivery systems, we hypothesize that by employing many of the clinical approaches from synthetic drug therapies to this new regenerative therapeutic tool, the efficacy of stem cell-based therapies can also be improved.
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Affiliation(s)
| | - Thamil Selvee Ramasamy
- />Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
| | - Noor Hayaty Abu Kasim
- />Department of Restorative Dentistry, Faculty of Dentistry, University of Malaya, Kuala Lumpur, Malaysia
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Anderson JJ, Boone JJ, Hansen M, Brady C, Gough A, Swayzee Z. Ankle arthrodesis fusion rates for mesenchymal stem cell bone allograft versus proximal tibia autograft. J Foot Ankle Surg 2014; 53:683-6. [PMID: 25158608 DOI: 10.1053/j.jfas.2014.06.029] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/01/2012] [Indexed: 02/03/2023]
Abstract
Ankle arthrodesis is commonly used in the treatment of ankle arthritis. The present study compared mesenchymal stem cell (MSC) bone allografts and proximal tibia autografts as adjuncts in performing ankle arthrodesis. A total of 109 consecutive ankle fusions performed from 2002 to 2008 were evaluated retrospectively. Of the 109 fusions, 24 were excluded from the present study, leaving 85 patients who had undergone ankle arthrodesis. Of the 85 patients, 41 had received a proximal tibia autograft and 44, an MSC bone allograft. These 2 groups were reviewed and compared retrospectively at least 2 years postoperatively for the overall fusion rate, interval to radiographic fusion, and interval to clinical fusion. A modified and adjusted American College of Foot and Ankle Surgeons ankle scale was used to measure patient satisfaction. The overall fusion rate was 84.1% in the MSC bone allograft group and 95.1% in the proximal tibia autograft group (p = .158). The corresponding mean intervals to radiographic fusion were 13.0 ± 2.5 weeks and 11.3 ± 2.8 weeks (p ≤ .001). The interval to clinical fusion was 13.1 ± 2.1 weeks and 11.0 ± 1.5 weeks (p ≤ .001) in the MSC bone allograft and proximal tibia autograft group, respectively. No statistically significant difference was found in the fusion rates between the MSC bone allograft and proximal tibia autograft groups. Also, no statistically significant difference was found between the preoperative and postoperative scores using a modified and adjusted American College of Foot and Ankle Surgeons ankle scale between the 2 groups (p = .41 and p = .44, respectively). A statistically significant delay to radiographic and clinical fusion was present in the MSC bone allograft group compared with the proximal tibia autograft group; however, no difference was found in patient satisfaction.
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Affiliation(s)
| | - Joshua J Boone
- Scripps Mercy-Kaiser San Diego Residency Program, San Diego, CA
| | | | - Chad Brady
- New Mexico Foot and Ankle Institute, Albuquerque, NM
| | - Adam Gough
- Gila Regional Medical Center, Silver City, NM
| | - Zflan Swayzee
- New Mexico Bone and Joint Institute, Alamogordo, NM.
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Bone regeneration in a massive rat femur defect through endochondral ossification achieved with chondrogenically differentiated MSCs in a degradable scaffold. Biomaterials 2014; 35:7800-10. [PMID: 24952976 DOI: 10.1016/j.biomaterials.2014.05.052] [Citation(s) in RCA: 119] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2014] [Accepted: 05/20/2014] [Indexed: 12/20/2022]
Abstract
Mesenchymal stem cells (MSCs) are multipotent cells capable of proliferating and differentiating into several lineages. In regenerative medicine, their potential as a resource for tissue-replacement therapy is receiving much attention. However, transplanting MSCs to repair larger bone defects in animal models has so far proved disappointing. Here we report on the healing of both critical-sized (5 mm) and massive (15 mm) full-thickness femur defects in rats by implanting a uniquely fabricated PLGA scaffold seeded with MSCs pre-differentiated in vitro into cartilage-forming chondrocytes (MSC-DCs). This strategy closely mimics endochondral ossification, the process by which long bones develop in nature. It is thought that because the transplanted MSC-DCs induced natural bone formation, the defect size was not critical to the outcome. Crucially, after 8 weeks the mean biomechanical strength of femora with the massive 15 mm implant reached 75% that of a normal rat femur, while in the case of 5 mm implants there was no significant difference. Successful healing was also highly reproducible, with bone union occurring in all treated animals examined radiologically 8 or 16 weeks after surgery.
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Rutledge K, Cheng Q, Pryzhkova M, Harris GM, Jabbarzadeh E. Enhanced differentiation of human embryonic stem cells on extracellular matrix-containing osteomimetic scaffolds for bone tissue engineering. Tissue Eng Part C Methods 2014; 20:865-74. [PMID: 24634988 DOI: 10.1089/ten.tec.2013.0411] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
Current methods of treating critical size bone defects include autografts and allografts, however, both present major limitations including donor-site morbidity, risk of disease transmission, and immune rejection. Tissue engineering provides a promising alternative to circumvent these shortcomings through the use of autologous cells, three-dimensional scaffolds, and growth factors. We investigated the development of a scaffold with native bone extracellular matrix (ECM) components for directing the osteogenic differentiation of human embryonic stem cells (hESCs). Toward this goal, a microsphere-sintering technique was used to fabricate poly(lactic-co-glycolic acid) (PLGA) scaffolds with optimum mechanical and structural properties. Human osteoblasts (hOBs) were seeded on these scaffolds to deposit bone ECM for 14 days. This was followed by a decellularization step leaving the mineralized matrix intact. Characterization of the decellularized PLGA scaffolds confirmed the deposition of calcium, collagen II, and alkaline phosphatase by osteoblasts. hESCs were seeded on the osteomimetic substrates in the presence of osteogenic growth medium, and osteogenicity was determined according to calcium content, osteocalcin expression, and bone marker gene regulation. Cell proliferation studies showed a constant increase in number for hESCs seeded on both PLGA and ECM-coated PLGA scaffolds. Calcium deposition by hESCs was significantly higher on the osteomimetic scaffolds compared with the control groups. Consistently, immunofluorescence staining demonstrated an increased expression of osteocalcin in hESCs seeded on ECM-coated osteomimetic PLGA scaffolds. Gene expression analysis of RUNX2 and osteocalcin further confirmed osteogenic differentiation of hESCs at the highest expression level on osteomimetic PLGA. These results together demonstrate the potential of PLGA scaffolds with native bone ECM components to direct osteogenic differentiation of hESCs and induce bone formation.
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Affiliation(s)
- Katy Rutledge
- 1 Department of Chemical Engineering, University of South Carolina , Columbia, South Carolina
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Miramond T, Corre P, Borget P, Moreau F, Guicheux J, Daculsi G, Weiss P. Osteoinduction of biphasic calcium phosphate scaffolds in a nude mouse model. J Biomater Appl 2014; 29:595-604. [DOI: 10.1177/0885328214537859] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
Bioceramics combined with isolated stem cells, or with total bone marrow, constitute the main strategies under consideration in the field of bone tissue engineering. In the present preclinical study, two biphasic calcium phosphate scaffolds currently on the market, MBCP® and MBCP+®, with different hydroxyapatite/β-tricalcium phosphate ratio, were implanted ectopically in a nude mouse model. These scaffolds were supplemented either with human mesenchymal stromal cells, or with human total bone marrow, or rat total bone marrow. Biomaterials alone were found to have potentially low, but non-zero, osteoinductive properties, while biomaterials associated with total bone marrow consistently improved osteoinduction in comparison with high concentrations of isolated human stromal cells.
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Affiliation(s)
- T Miramond
- INSERM UMRS 791, University of Nantes, Nantes, France
- Biomatlante SA, Vigneux-de-Bretagne, France
| | - P Corre
- INSERM UMRS 791, University of Nantes, Nantes, France
- Departments of Stomatology and Maxillofacial Surgery, Nantes University Hospital, Nantes, France
| | - P Borget
- Biomatlante SA, Vigneux-de-Bretagne, France
| | - F Moreau
- Biomatlante SA, Vigneux-de-Bretagne, France
| | - J Guicheux
- INSERM UMRS 791, University of Nantes, Nantes, France
| | - G Daculsi
- INSERM UMRS 791, University of Nantes, Nantes, France
| | - P Weiss
- INSERM UMRS 791, University of Nantes, Nantes, France
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46
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The role of mesenchymal stem cells in bone repair and regeneration. EUROPEAN JOURNAL OF ORTHOPAEDIC SURGERY AND TRAUMATOLOGY 2013; 24:257-62. [DOI: 10.1007/s00590-013-1328-5] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/12/2013] [Accepted: 09/24/2013] [Indexed: 12/13/2022]
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Kuhn LT, Liu Y, Boyd NL, Dennis JE, Jiang X, Xin X, Charles LF, Wang L, Aguila HL, Rowe DW, Lichtler AC, Goldberg AJ. Developmental-like bone regeneration by human embryonic stem cell-derived mesenchymal cells. Tissue Eng Part A 2013; 20:365-77. [PMID: 23952622 DOI: 10.1089/ten.tea.2013.0321] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022] Open
Abstract
The in vivo osteogenesis potential of mesenchymal-like cells derived from human embryonic stem cells (hESC-MCs) was evaluated in vivo by implantation on collagen/hydroxyapatite scaffolds into calvarial defects in immunodeficient mice. This study is novel because no osteogenic or chondrogenic differentiation protocols were applied to the cells prior to implantation. After 6 weeks, X-ray, microCT, and histological analysis showed that the hESC-MCs had consistently formed a highly vascularized new bone that bridged the bone defect and seamlessly integrated with host bone. The implanted hESC-MCs differentiated in situ to functional hypertrophic chondrocytes, osteoblasts, and osteocytes forming new bone tissue via an endochondral ossification pathway. Evidence for the direct participation of the human cells in bone morphogenesis was verified by two separate assays: with Alu and by human mitochondrial antigen positive staining in conjunction with co-localized expression of human bone sialoprotein in histologically verified regions of new bone. The large volume of new bone in a calvarial defect and the direct participation of the hESC-MCs far exceeds that of previous studies and that of the control adult hMSCs. This study represents a key step forward for bone tissue engineering because of the large volume, vascularity, and reproducibility of new bone formation and the discovery that it is advantageous to not over-commit these progenitor cells to a particular lineage prior to implantation. The hESC-MCs were able to recapitulate the mesenchymal developmental pathway and were able to repair the bone defect semi-autonomously without preimplantation differentiation to osteo- or chondroprogenitors.
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Affiliation(s)
- Liisa T Kuhn
- 1 Department of Reconstructive Sciences, Center for Biomaterials, School of Dental Medicine, University of Connecticut Health Center , Farmington, Connecticut
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48
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Jungbluth P, Hakimi AR, Grassmann JP, Schneppendahl J, Betsch M, Kröpil P, Thelen S, Sager M, Herten M, Wild M, Windolf J, Hakimi M. The early phase influence of bone marrow concentrate on metaphyseal bone healing. Injury 2013; 44:1285-94. [PMID: 23684350 DOI: 10.1016/j.injury.2013.04.015] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/21/2013] [Revised: 03/30/2013] [Accepted: 04/14/2013] [Indexed: 02/02/2023]
Abstract
Bone marrow concentrate (BMC) contains high densities of progenitor cells. Therefore, in critical size defects BMC may have the potency to support bone healing. The aim of this study was to investigate the effect of BMC in combination with calcium phosphate granules (CPG) on bone defect healing in a metaphyseal long bone defect in mini-pigs. A metaphyseal critical-size bone defect at the proximal tibia of 24 mini-pigs was filled with CPG combined with BMC, CPG solely (control group) or with an autograft. Radiological and histomorphometrical evaluations after 6 weeks (42 days) showed significantly more bone formation in the BMC group in the central area of the defect zone and the cortical defect zone compared to the CPG group. At the same time the resorption rate of CPG increased significantly in the BMC group. Nevertheless, compared to the BMC group the autograft group showed a significantly higher new bone formation radiologically and histomorphometrically. In BMC the count of mononuclear cells was significantly higher compared to the bone marrow aspirate (3.5-fold). The mesenchymal progenitor cell characteristics of the cells in BMC were confirmed by flow cytometry. Cells from BMC created significantly larger colonies of alkaline phosphatase-positive colony forming units (CFU-ALP) (4.4-fold) compared to cells from bone marrow aspirate. Nevertheless, even in the BMC group complete osseous bridging was only detectable in isolated instances of the bone defects. Within the limitations of this study the BMC+CPG composite promotes bone regeneration in the early phase of bone healing significantly better than the isolated application of CPG. However, the addition of BMC does not lead to a solid fusion of the defect in the early phase of bone healing an still does not represent an equal alternative to autologous bone.
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Affiliation(s)
- P Jungbluth
- Heinrich Heine University Hospital Duesseldorf, Department of Trauma and Handsurgery, Moorenstr. 5, 40225 Duesseldorf, Germany
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Veronesi E, Murgia A, Caselli A, Grisendi G, Piccinno MS, Rasini V, Giordano R, Montemurro T, Bourin P, Sensebé L, Rojewski MT, Schrezenmeier H, Layrolle P, Ginebra MP, Panaitescu CB, Gómez-Barrena E, Catani F, Paolucci P, Burns JS, Dominici M. Transportation conditions for prompt use of ex vivo expanded and freshly harvested clinical-grade bone marrow mesenchymal stromal/stem cells for bone regeneration. Tissue Eng Part C Methods 2013; 20:239-51. [PMID: 23845029 DOI: 10.1089/ten.tec.2013.0250] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
Abstract
Successful preliminary studies have encouraged a more translational phase for stem cell research. Nevertheless, advances in the culture of human bone marrow-derived mesenchymal stromal/stem cells (hBM-MSC) and osteoconductive qualities of combined biomaterials can be undermined if necessary cell transportation procedures prove unviable. We aimed at evaluating the effect of transportation conditions on cell function, including the ability to form bone in vivo, using procedures suited to clinical application. hBM-MSC expanded in current Good Manufacturing Practice (cGMP) facilities (cGMP-hBM-MSC) to numbers suitable for therapy were transported overnight within syringes and subsequently tested for viability. Scaled-down experiments mimicking shipment for 18 h at 4°C tested the influence of three different clinical-grade transportation buffers (0.9% saline alone or with 4% human serum albumin [HSA] from two independent sources) compared with cell maintenance medium. Cell viability after shipment was >80% in all cases, enabling evaluation of (1) adhesion to plastic flasks and hydroxyapatite tricalcium phosphate osteoconductive biomaterial (HA/β-TCP 3D scaffold); (2) proliferation rate; (3) ex vivo osteogenic differentiation in contexts of 2D monolayers on plastic and 3D HA/β-TCP scaffolds; and (4) in vivo ectopic bone formation after subcutaneous implantation of cells with HA/β-TCP scaffold into NOD/SCID mice. Von Kossa staining was used to assess ex vivo osteogenic differentiation in 3D cultures, providing a quantifiable test of 3D biomineralization ex vivo as a rapid, cost-effective potency assay. Near-equivalent capacities for cell survival, proliferation, and osteogenic differentiation were found for all transportation buffers. Moreover, cGMP-hBM-MSC transported from a production facility under clinical-grade conditions of 4% HSA in 0.9% saline to a destination 18 h away showed prompt adhesion to HA/β-TCP 3D scaffold and subsequent in vivo bone formation. A successfully validated transportation protocol extends the applicability of fresh stem cells involving multicentric trials for regenerative medicine.
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Affiliation(s)
- Elena Veronesi
- 1 Laboratory of Cell Biology and Advanced Cancer Therapies, Department of Medical and Surgical Sciences for Children & Adults, University Hospital of Modena and Reggio Emilia , Modena, Italy
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50
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Brohem CA, de Carvalho CM, Radoski CL, Santi FC, Baptista MC, Swinka BB, de A. Urban C, de Araujo LRR, Graf RM, Feferman IHS, Lorencini M. Comparison between fibroblasts and mesenchymal stem cells derived from dermal and adipose tissue. Int J Cosmet Sci 2013; 35:448-57. [DOI: 10.1111/ics.12064] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2013] [Accepted: 04/28/2013] [Indexed: 12/11/2022]
Affiliation(s)
- C. A. Brohem
- Department of Research and Development; Grupo Boticário; Biomolecular Research Laboratory; São José dos Pinhais; Paraná; Brazil
| | | | - C. L. Radoski
- Department of Biotechnology; Positivo University; Curitiba; Paraná; Brazil
| | - F. C. Santi
- Department of Research and Development; Grupo Boticário; Biomolecular Research Laboratory; São José dos Pinhais; Paraná; Brazil
| | - M. C. Baptista
- Department of Research and Development; Grupo Boticário; Biomolecular Research Laboratory; São José dos Pinhais; Paraná; Brazil
| | - B. B. Swinka
- Department of Research and Development; Grupo Boticário; Biomolecular Research Laboratory; São José dos Pinhais; Paraná; Brazil
| | - C. de A. Urban
- Department of Biotechnology; Positivo University; Curitiba; Paraná; Brazil
| | | | - R. M. Graf
- Department of Plastic Surgery; Federal University of Paraná; Curitiba; Paraná; Brazil
| | - I. H. S. Feferman
- Department of Research and Development; Grupo Boticário; Biomolecular Research Laboratory; São José dos Pinhais; Paraná; Brazil
| | - M. Lorencini
- Department of Research and Development; Grupo Boticário; Biomolecular Research Laboratory; São José dos Pinhais; Paraná; Brazil
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