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A Comparative Study on the Adipogenic Differentiation of Mesenchymal Stem/Stromal Cells in 2D and 3D Culture. Cells 2022; 11:cells11081313. [PMID: 35455993 PMCID: PMC9029885 DOI: 10.3390/cells11081313] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2022] [Revised: 04/07/2022] [Accepted: 04/08/2022] [Indexed: 02/04/2023] Open
Abstract
Mesenchymal stem/stromal cells (MSC) are capable of renewing the progenitor cell fraction or differentiating in a tissue-specific manner. Adipogenic differentiation of adipose-tissue-derived MSC (adMSC) is important in various pathological processes. Adipocytes and their progenitors are metabolically active and secrete molecules (adipokines) that have both pro- and anti-inflammatory properties. Cell culturing in 2D is commonly used to study cellular responses, but the 2D environment does not reflect the structural situation for most cell types. Therefore, 3D culture systems have been developed to create an environment considered more physiological. Since knowledge about the effects of 3D cultivation on adipogenic differentiation is limited, we investigated its effects on adipogenic differentiation and adipokine release of adMSC (up to 28 days) and compared these with the effects in 2D. We demonstrated that cultivation conditions are crucial for cell behavior: in both 2D and 3D culture, adipogenic differentiation occurred only after specific stimulation. While the size and structure of adipogenically stimulated 3D spheroids remained stable during the experiment, the unstimulated spheroids showed signs of disintegration. Adipokine release was dependent on culture dimensionality; we found upregulated adiponectin and downregulated pro-inflammatory factors. Our findings are relevant for cell therapeutic applications of adMSC in complex, three-dimensionally arranged tissues.
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Araszkiewicz AM, Oliveira EP, Svendsen T, Drela K, Rogujski P, Malysz-Cymborska I, Fiedorowicz M, Reis RL, Oliveira JM, Walczak P, Janowski M, Lukomska B, Stanaszek L. Manganese-Labeled Alginate Hydrogels for Image-Guided Cell Transplantation. Int J Mol Sci 2022; 23:ijms23052465. [PMID: 35269609 PMCID: PMC8910205 DOI: 10.3390/ijms23052465] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2021] [Revised: 02/18/2022] [Accepted: 02/18/2022] [Indexed: 02/04/2023] Open
Abstract
Cell transplantation has been studied extensively as a therapeutic strategy for neurological disorders. However, to date, its effectiveness remains unsatisfactory due to low precision and efficacy of cell delivery; poor survival of transplanted cells; and inadequate monitoring of their fate in vivo. Fortunately, different bio-scaffolds have been proposed as cell carriers to improve the accuracy of cell delivery, survival, differentiation, and controlled release of embedded stem cells. The goal of our study was to establish hydrogel scaffolds suitable for stem cell delivery that also allow non-invasive magnetic resonance imaging (MRI). We focused on alginate-based hydrogels due to their natural origin, biocompatibility, resemblance to the extracellular matrix, and easy manipulation of gelation processes. We optimized the properties of alginate-based hydrogels, turning them into suitable carriers for transplanted cells. Human adipose-derived stem cells embedded in these hydrogels survived for at least 14 days in vitro. Alginate-based hydrogels were also modified successfully to allow their injectability via a needle. Finally, supplementing alginate hydrogels with Mn ions or Mn nanoparticles allowed for their visualization in vivo using manganese-enhanced MRI. We demonstrated that modified alginate-based hydrogels can support therapeutic cells as MRI-detectable matrices.
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Affiliation(s)
- Antonina M. Araszkiewicz
- NeuroRepair Department, Mossakowski Medical Research Institute, Polish Academy of Sciences, 02-106 Warsaw, Poland; (A.M.A.); (P.R.); (B.L.)
| | - Eduarda P. Oliveira
- 3B’s Research Group, I3Bs—Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, 4805-017 Guimarães, Portugal; (E.P.O.); (R.L.R.); (J.M.O.)
- ICVS/3B’s—PT Government Associated Laboratory, 4710-057 Guimarães, Portugal
| | | | | | - Piotr Rogujski
- NeuroRepair Department, Mossakowski Medical Research Institute, Polish Academy of Sciences, 02-106 Warsaw, Poland; (A.M.A.); (P.R.); (B.L.)
| | - Izabela Malysz-Cymborska
- Department of Neurosurgery, School of Medicine, Collegium Medicum, University of Warmia and Mazury, 10-082 Olsztyn, Poland;
| | - Michal Fiedorowicz
- Small Animal Magnetic Resonance Imaging Laboratory, Mossakowski Medical Research Institute, Polish Academy of Sciences, 02-106 Warsaw, Poland;
| | - Rui L. Reis
- 3B’s Research Group, I3Bs—Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, 4805-017 Guimarães, Portugal; (E.P.O.); (R.L.R.); (J.M.O.)
- ICVS/3B’s—PT Government Associated Laboratory, 4710-057 Guimarães, Portugal
| | - Joaquim Miguel Oliveira
- 3B’s Research Group, I3Bs—Research Institute on Biomaterials, Biodegradables and Biomimetics, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, 4805-017 Guimarães, Portugal; (E.P.O.); (R.L.R.); (J.M.O.)
- ICVS/3B’s—PT Government Associated Laboratory, 4710-057 Guimarães, Portugal
| | - Piotr Walczak
- Program for Image Guided Neurointerventions, Department of Diagnostic Radiology and Nuclear Medicine, Center for Advanced Imaging Research, University of Maryland Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland, Baltimore, MD 21201, USA; (P.W.); (M.J.)
| | - Miroslaw Janowski
- Program for Image Guided Neurointerventions, Department of Diagnostic Radiology and Nuclear Medicine, Center for Advanced Imaging Research, University of Maryland Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland, Baltimore, MD 21201, USA; (P.W.); (M.J.)
| | - Barbara Lukomska
- NeuroRepair Department, Mossakowski Medical Research Institute, Polish Academy of Sciences, 02-106 Warsaw, Poland; (A.M.A.); (P.R.); (B.L.)
| | - Luiza Stanaszek
- NeuroRepair Department, Mossakowski Medical Research Institute, Polish Academy of Sciences, 02-106 Warsaw, Poland; (A.M.A.); (P.R.); (B.L.)
- Correspondence: ; Tel.: +48-226-086-529
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Gibler P, Gimble J, Hamel K, Rogers E, Henderson M, Wu X, Olesky S, Frazier T. Human Adipose-Derived Stromal/Stem Cell Culture and Analysis Methods for Adipose Tissue Modeling In Vitro: A Systematic Review. Cells 2021; 10:1378. [PMID: 34204869 PMCID: PMC8227575 DOI: 10.3390/cells10061378] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2021] [Revised: 05/25/2021] [Accepted: 05/26/2021] [Indexed: 12/11/2022] Open
Abstract
Human adipose-derived stromal/stem cells (hASC) are widely used for in vitro modeling of physiologically relevant human adipose tissue. These models are useful for the development of tissue constructs for soft tissue regeneration and 3-dimensional (3D) microphysiological systems (MPS) for drug discovery. In this systematic review, we report on the current state of hASC culture and assessment methods for adipose tissue engineering using 3D MPS. Our search efforts resulted in the identification of 184 independent records, of which 27 were determined to be most relevant to the goals of the present review. Our results demonstrate a lack of consensus on methods for hASC culture and assessment for the production of physiologically relevant in vitro models of human adipose tissue. Few studies have assessed the impact of different 3D culture conditions on hASC adipogenesis. Additionally, there has been a limited use of assays for characterizing the functionality of adipose tissue in vitro. Results from this study suggest the need for more standardized culture methods and further analysis on in vitro tissue functionality. These will be necessary to validate the utility of 3D MPS as an in vitro model to reduce, refine, and replace in vivo experiments in the drug discovery regulatory process.
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Affiliation(s)
- Peyton Gibler
- Obatala Sciences Inc., New Orleans, LA 70148, USA; (P.G.); (K.H.); (E.R.); (M.H.); (X.W.); (S.O.); (T.F.)
| | - Jeffrey Gimble
- Obatala Sciences Inc., New Orleans, LA 70148, USA; (P.G.); (K.H.); (E.R.); (M.H.); (X.W.); (S.O.); (T.F.)
- Department of Structural and Cell Biology, Tulane University School of Medicine, New Orleans, LA 70112, USA
- Department of Medicine, Tulane University School of Medicine, New Orleans, LA 70112, USA
- Department of Surgery, Tulane University School of Medicine, New Orleans, LA 70112, USA
| | - Katie Hamel
- Obatala Sciences Inc., New Orleans, LA 70148, USA; (P.G.); (K.H.); (E.R.); (M.H.); (X.W.); (S.O.); (T.F.)
| | - Emma Rogers
- Obatala Sciences Inc., New Orleans, LA 70148, USA; (P.G.); (K.H.); (E.R.); (M.H.); (X.W.); (S.O.); (T.F.)
| | - Michael Henderson
- Obatala Sciences Inc., New Orleans, LA 70148, USA; (P.G.); (K.H.); (E.R.); (M.H.); (X.W.); (S.O.); (T.F.)
| | - Xiying Wu
- Obatala Sciences Inc., New Orleans, LA 70148, USA; (P.G.); (K.H.); (E.R.); (M.H.); (X.W.); (S.O.); (T.F.)
| | - Spencer Olesky
- Obatala Sciences Inc., New Orleans, LA 70148, USA; (P.G.); (K.H.); (E.R.); (M.H.); (X.W.); (S.O.); (T.F.)
| | - Trivia Frazier
- Obatala Sciences Inc., New Orleans, LA 70148, USA; (P.G.); (K.H.); (E.R.); (M.H.); (X.W.); (S.O.); (T.F.)
- Department of Structural and Cell Biology, Tulane University School of Medicine, New Orleans, LA 70112, USA
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Bijonowski BM, Yuan X, Jeske R, Li Y, Grant SC. Cyclical aggregation extends in vitro expansion potential of human mesenchymal stem cells. Sci Rep 2020; 10:20448. [PMID: 33235227 PMCID: PMC7686385 DOI: 10.1038/s41598-020-77288-4] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2020] [Accepted: 11/09/2020] [Indexed: 02/06/2023] Open
Abstract
Mesenchymal stem cell (MSC)-based therapy has shown great promises in various animal disease models. However, this therapeutic potency has not been well claimed when applied to human clinical trials. This is due to both the availability of MSCs at the time of administration and lack of viable expansion strategies. MSCs are very susceptible to in vitro culture environment and tend to adapt the microenvironment which could lead to cellular senescence and aging. Therefore, extended in vitro expansion induces loss of MSC functionality and its clinical relevance. To combat this effect, this work assessed a novel cyclical aggregation as a means of expanding MSCs to maintain stem cell functionality. The cyclical aggregation consists of an aggregation phase and an expansion phase by replating the dissociated MSC aggregates onto planar tissue culture surfaces. The results indicate that cyclical aggregation maintains proliferative capability, stem cell proteins, and clonogenicity, and prevents the acquisition of senescence. To determine why aggregation was responsible for this phenomenon, the integrated stress response pathway was probed with salubrial and GSK-2606414. Treatment with salubrial had no significant effect, while GSK-2606414 mitigated the effects of aggregation leading to in vitro aging. This method holds the potential to increase the clinical relevance of MSC therapeutic effects from small model systems (such as rats and mice) to humans, and may open the potential of patient-derived MSCs for treatment thereby removing the need for immunosuppression.
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Affiliation(s)
- Brent M Bijonowski
- Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University, 2525 Pottsdamer St., Tallahassee, FL, 32310, USA.
- University of Münster, Münster, Germany.
| | - Xuegang Yuan
- Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University, 2525 Pottsdamer St., Tallahassee, FL, 32310, USA
- The National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL, USA
| | - Richard Jeske
- Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University, 2525 Pottsdamer St., Tallahassee, FL, 32310, USA
| | - Yan Li
- Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University, 2525 Pottsdamer St., Tallahassee, FL, 32310, USA.
| | - Samuel C Grant
- Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University, 2525 Pottsdamer St., Tallahassee, FL, 32310, USA
- The National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL, USA
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Baptista LS. Adipose stromal/stem cells in regenerative medicine: Potentials and limitations. World J Stem Cells 2020; 12:1-7. [PMID: 32110271 PMCID: PMC7031762 DOI: 10.4252/wjsc.v12.i1.1] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/03/2019] [Revised: 10/11/2019] [Accepted: 11/14/2019] [Indexed: 02/07/2023] Open
Abstract
This article presents the stem and progenitor cells from subcutaneous adipose tissue, briefly comparing them with their bone marrow counterparts, and discussing their potential for use in regenerative medicine. Subcutaneous adipose tissue differs from other mesenchymal stromal/stem cells (MSCs) sources in that it contains a pre-adipocyte population that dwells in the adventitia of robust blood vessels. Pre-adipocytes are present both in the stromal-vascular fraction (SVF; freshly isolated cells) and in the adherent fraction of adipose stromal/stem cells (ASCs; in vitro expanded cells), and have an active role on the chronic inflammation environment established in obesity, likely due their monocytic-macrophage lineage identity. The SVF and ASCs have been explored in cell therapy protocols with relative success, given their paracrine and immunomodulatory effects. Importantly, the widely explored multipotentiality of ASCs has direct application in bone, cartilage and adipose tissue engineering. The aim of this editorial is to reinforce the peculiarities of the stem and progenitor cells from subcutaneous adipose tissue, revealing the spheroids as a recently described biotechnological tool for cell therapy and tissue engineering. Innovative cell culture techniques, in particular 3D scaffold-free cultures such as spheroids, are now available to increase the potential for regeneration and differentiation of mesenchymal lineages. Spheroids are being explored not only as a model for cell differentiation, but also as powerful 3D cell culture tools to maintain the stemness and expand the regenerative and differentiation capacities of mesenchymal cell lineages.
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Affiliation(s)
- Leandra Santos Baptista
- Multidisciplinary Center for Biological Research (Numpex-Bio), Federal University of Rio de Janeiro (UFRJ) Campus Duque de Caxias, Duque de Caxias, RJ 25245-390, Brazil
- Post-graduate Program in Biotechnology, National Institute of Metrology, Quality and Technology (INMETRO), Duque de Caxias, RJ 25250-020, Brazil
- Post-graduate Program in Translational Biomedicine (Biotrans), Unigranrio, Campus I, Duque de Caxias, Duque de Caxias, RJ 25250-020, Brazil
- Laboratory of Tissue Bioengineering, Directory of Metrology Applied to Life Sciences, National Institute of Metrology, Quality and Technology (INMETRO), Duque de Caxias, RJ 25250-020, Brazil
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Experimental Strategies of Mesenchymal Stem Cell Propagation: Adverse Events and Potential Risk of Functional Changes. Stem Cells Int 2019; 2019:7012692. [PMID: 30956673 PMCID: PMC6431404 DOI: 10.1155/2019/7012692] [Citation(s) in RCA: 80] [Impact Index Per Article: 13.3] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2018] [Revised: 12/28/2018] [Accepted: 01/13/2019] [Indexed: 12/16/2022] Open
Abstract
Mesenchymal stem cells (MSCs) are attractive candidates for cell-based tissue repair approaches. Hundreds of clinical trials using MSCs have been completed and many others are still being investigated. For most therapeutic applications, MSC propagation in vitro is often required. However, ex vivo culture condition is not fully physiological and may affect biological properties of MSCs including their regenerative potential. Moreover, both cell cryopreservation and labelling procedure prior to infusion may have the negative impact on their expected effect in vivo. The incidence of MSC transformation during in vitro culture should be also taken into consideration before using cells in stem cell therapy. In our review, we focused on different aspects of MSC propagation that might influence their regenerative properties of MSC. We also discussed the influence of different factors that might abolish MSC proliferation and differentiation as well as potential impact of stem cell senescence and aging. Despite of many positive therapeutic effects of MSC therapy, one has to be conscious about potential cell changes that could appear during manufacturing of MSCs.
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Yipeng J, Yongde X, Yuanyi W, Jilei S, Jiaxiang G, Jiangping G, Yong Y. Microtissues Enhance Smooth Muscle Differentiation and Cell Viability of hADSCs for Three Dimensional Bioprinting. Front Physiol 2017; 8:534. [PMID: 28790931 PMCID: PMC5524823 DOI: 10.3389/fphys.2017.00534] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2017] [Accepted: 07/10/2017] [Indexed: 12/22/2022] Open
Abstract
Smooth muscle differentiated human adipose derived stem cells (hADSCs) provide a crucial stem cell source for urinary tissue engineering, but the induction of hADSCs for smooth muscle differentiation still has several issues to overcome, including a relatively long induction time and equipment dependence, which limits access to abundant stem cells within a short period of time for further application. Three-dimensional (3D) bioprinting holds great promise in regenerative medicine due to its controllable construction of a designed 3D structure. When evenly mixed with bioink, stem cells can be spatially distributed within a bioprinted 3D structure, thus avoiding drawbacks such as, stem cell detachment in a conventional cell-scaffold strategy. Notwithstanding the advantages mentioned above, cell viability is often compromised during 3D bioprinting, which is often due to pressure during the bioprinting process. The objective of our study was to improve the efficiency of hADSC smooth muscle differentiation and cell viability of a 3D bioprinted structure. Here, we employed the hanging-drop method to generate hADSC microtissues in a smooth muscle inductive medium containing human transforming growth factor β1 and bioprinted the induced microtissues onto a 3D structure. After 3 days of smooth muscle induction, the expression of α-smooth muscle actin and smoothelin was higher in microtissues than in their counterpart monolayer cultured hADSCs, as confirmed by immunofluorescence and western blotting analysis. The semi-quantitative assay showed that the expression of α-smooth muscle actin (α-SMA) was 0.218 ± 0.077 in MTs and 0.082 ± 0.007 in Controls; smoothelin expression was 0.319 ± 0.02 in MTs and 0.178 ± 0.06 in Controls. Induced MTs maintained their phenotype after the bioprinting process. Live/dead and cell count kit 8 assays showed that cell viability and cell proliferation in the 3D structure printed with microtissues were higher at all time points compared to the conventional single-cell bioprinting strategy (mean cell viability was 88.16 ± 3.98 vs. 61.76 ± 15% for microtissues and single-cells, respectively). These results provide a novel way to enhance the smooth muscle differentiation of hADSCs and a simple method to maintain better cell viability in 3D bioprinting.
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Affiliation(s)
- Jin Yipeng
- Department of Urology, Chinese PLA General HospitalBeijing, China
| | - Xu Yongde
- Department of Urology, First Affiliated Hospital of Chinese PLA General HospitalBeijing, China
| | - Wu Yuanyi
- Department of Urology, First Affiliated Hospital of Chinese PLA General HospitalBeijing, China
| | - Sun Jilei
- Department of Urology, First Affiliated Hospital of Chinese PLA General HospitalBeijing, China
| | - Guo Jiaxiang
- Department of Urology, First Affiliated Hospital of Chinese PLA General HospitalBeijing, China
| | - Gao Jiangping
- Department of Urology, Chinese PLA General HospitalBeijing, China
| | - Yang Yong
- Department of Urology, First Affiliated Hospital of Chinese PLA General HospitalBeijing, China
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Zhou Q, Li B, Zhao J, Pan W, Xu J, Chen S. IGF-I induces adipose derived mesenchymal cell chondrogenic differentiation in vitro and enhances chondrogenesis in vivo. In Vitro Cell Dev Biol Anim 2016; 52:356-364. [PMID: 26822434 DOI: 10.1007/s11626-015-9969-9] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2015] [Accepted: 10/16/2015] [Indexed: 12/29/2022]
Abstract
Recent studies have demonstrated that insulin-like growth factor-1 (IGF-I) modulates bone mesenchymal stem cell chondrogenic differentiation independent of transforming growth factor beta (TGF-β) signaling in vitro. However, it is unclear whether IGF-I can solely modulate human adipose-derived mesenchymal cell (hAMC) chondrogenic differentiation, or whether it has additive effects with TGF-β1 to induce chondrogenic differentiation in vitro and development of mature cartilage in vivo. We investigated the effect of IGF-I on the induction of hAMC chondrogenic differentiation in the presence or absence of transforming growth factor beta 1 (TGF-β1) in vitro, and chondrogenesis of the induced hAMC in vivo. The results showed that IGF-I alone induced collagen type II, aggrecan, and Sox9 mRNA expression and collagen type II and aggrecan proteins expressions in hAMCs. Notably, there was greater mRNA expression of collagen type II, aggrecan and Sox9, and greater protein expression of collagen type II and aggrecan following TGF-β1 + IGF-I treatment, compared to either TGF-β1 or IGF-I-treated hAMCs. These results were confirmed in cartilage tissues derived from induced hAMCs. These findings indicate that IGF-I alone has the ability to induce chondrogenic differentiation and has additive effects with TGF-β1 to induce chondrogenic differentiation in vitro and in vivo.
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Affiliation(s)
- Quan Zhou
- Department of Orthopaedics, Huai'an Hospital Affiliated of Xuzhou Medical College and Huai'an Second Hospital, No. 62 Huaihai Road South, Huai'an, 223002, China
| | - Baojun Li
- Department of Joint Surgery, Second People's Hospital of Hunan Province, Changsha, 410007, China
| | - Jiali Zhao
- Department of Orthopaedics, Huai'an Hospital Affiliated of Xuzhou Medical College and Huai'an Second Hospital, No. 62 Huaihai Road South, Huai'an, 223002, China
| | - Wei Pan
- Department of Orthopaedics, Huai'an Hospital Affiliated of Xuzhou Medical College and Huai'an Second Hospital, No. 62 Huaihai Road South, Huai'an, 223002, China
| | - Jin Xu
- Department of Orthopaedics, Huai'an Hospital Affiliated of Xuzhou Medical College and Huai'an Second Hospital, No. 62 Huaihai Road South, Huai'an, 223002, China
| | - Sumei Chen
- Department of Orthopaedics, Huai'an Hospital Affiliated of Xuzhou Medical College and Huai'an Second Hospital, No. 62 Huaihai Road South, Huai'an, 223002, China.
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