P- Reviewer: Campo SMA, Hokama A S- Editor: Ma YJ L- Editor: AmEditor E- Editor: Zhang DN
Published online Mar 21, 2015. doi: 10.3748/wjg.v21.i11.3330
Peer-review started: October 3, 2014
First decision: October 29, 2014
Revised: November 18, 2014
Accepted: January 16, 2015
Article in press: January 16, 2015
Published online: March 21, 2015
AIM: To study the results of stem-cell therapy under a Compassionate-use Program for patients with recurrent anal fistulae.
METHODS: Under controlled circumstances, and approved by European and Spanish laws, a Compassionate-use Program allows the use of stem-cell therapy for patients with very complex anal fistulae. Candidates had previously undergone multiple surgical interventions that had failed to resolve the fistulae, and presented symptomatic recurrence. The intervention consisted of limited surgery (with closure of the internal opening), followed by local implant of stem cells in the fistula-tract wall. Autologous expanded adipose-derived stem cells were the main cell type selected for implant. The first evaluation was performed on the 8th postoperative week; outcome was classified as response or partial response. Evaluation one year after the intervention confirmed if complete healing of the fistula was achieved.
RESULTS: Ten patients (8 male) with highly recurrent and complex fistulae were treated (mean age: 49 years, range: 28-76 years). Seven cases were non-Crohn’s fistulae, and three were Crohn’s-associated fistulae. Previous surgical attempts ranged from 3 to 12. Two patients presented with preoperative incontinence (Wexner scores of 12 and 13 points). After the intervention, six patients showed clinical response on the 8th postoperative week, with a complete cessation of suppuration from the fistula. Three patients presented a partial response, with an evident decrease in suppuration. A year later, six patients (60%) remained healed, with complete reepithelization of the external opening. Postoperative Wexner Scores were 0 in six cases. The two patients with previous incontinence improved their scores from 12 to 8 points and from 13 to 5 points. No adverse reactions or complications related to stem-cell therapy were reported during the study period.
CONCLUSION: Stem cells are safe and useful for treating anal fistulae. Healing can be achieved in severe cases, sparing fecal incontinence risk, and improving previous scoring.
Core tip: Our group has performed various clinical trials with adipose stem cells. Patients with very complex fistulae, multiple previous surgeries, and treatment failure are generally not able to enter these studies despite the benefit and “last chance” of cure. We present the results of a Compassionate-use Program, which enabled the application of stem-cell therapy to these patients, under strict regulations. Ten patients were treated, and after one year of follow-up, we conclude that adipose stem cells are effective and safe, and 60% of the patients achieved complete healing.
- Citation: Garcia-Olmo D, Guadalajara H, Rubio-Perez I, Herreros MD, de-la-Quintana P, Garcia-Arranz M. Recurrent anal fistulae: Limited surgery supported by stem cells. World J Gastroenterol 2015; 21(11): 3330-3336
- URL: https://www.wjgnet.com/1007-9327/full/v21/i11/3330.htm
- DOI: https://dx.doi.org/10.3748/wjg.v21.i11.3330
A limited surgical treatment in recurrent perianal fistulae often results in new recurrence, whereas there is a high risk of fecal incontinence if an extensive surgical treatment is performed[1-3]. The use of stem cells to treat complex fistulae is a promising area of research[4,5], for they may help to regenerate damaged perianal tissue. Especially in Crohn’s disease, the presence of these cells could favor healing through anti-inflammatory and immunomodulatory effects[6-9]. Various randomized controlled trials using stem cells for the treatment of anal fistulae have already been conducted, and all of them show an excellent safety profile. Nevertheless, the real efficacy is currently difficutl to assess. A recent Spanish study revealed that the mean annual global cost of conventional treatments for patients with Crohn’s disease and perianal fistulae is > € 8000/year.
According to current regulatory issues at the time (2002), our team started a clinical trial process in order to test the ability of adipose-derived stem cells (ASC) to improve healing in complex perianal fistulae, including those associated with Crohn’s disease. The chosen cell source was adipose tissue because the harvesting process for ASC following liposuction was simple and could be performed in our on-site laboratory. To the date, we have finished a complete clinical trial process: a pilot study, and Phase II and Phase III clinical trials. Although a complex perianal fistula is the worst scenario, we observed satisfactory healing in our patients, without associated fecal incontinence. We are currently developing novel clinical trials directed to test different strategies in order to improve our results.
Some of the patients with multi-recurrent anal fistulae did not meet the strict eligibility criteria of clinical trials or were scheduled in control groups. The only option to treat these fistulae with stem cells was by “compassionate use”. To achieve this, the European regulatory laws and the Spanish Medicine Agency guidelines were followed in order to obtain regulatory permissions. Under the Compassionate-use Program, the surgical technique and the cells’ lineage is tailored for each patient, reinforcing the possibilities of cure, as opposed to the clinical trial setting. In these special cases, we performed minimal surgical maneuvers (limited surgery) directed to the conditioning of the surgical field, followed by implant of cells, in order to improve healing. This strategy enabled us to avoid anal sphincter injury and facilitated cell homing.
The aim of this paper is to report our experience in a clinical trial- complementary Compassionate-use Program, and discuss the possible clinical uses of stem cells in the future, focusing on the treatment of complex and recurrent perianal fistulae.
We present an observational study, including 10 patients (8 male and 2 female) with recurrent perianal fistulae who had previously undergone at least three surgical interventions (maximum: 12, average: 6.2), with failure to resolve the fistula. The mean age of the patients was 49 years, and ranged from 28 to 76 years (Table 1). Seven patients presented complex non-Crohn’s fistulae (four were Parks’ type III) and three patients had Crohn’s-associated perianal fistulae. Two of these patients complained of fecal incontinence at the moment of enrollment in this study, with a Wexner Score > 10.
|ID||Sex||Age (yr)||Crohn’s disease||Park’s classification||Previous surgical attempts||Initial incontinence score (Wexner)||Surgical technique||Fibrin glue||Cells||Response 8th week||Incontinence score 8th week||Healing one year after|
|1||Male||58||No||III||8||1||Flap + deep curettage||No||eASC||Yes||0||Yes|
|2||Male||43||No||II||4||Unknown||IO closure + partial fistulectomy||Yes||eASC||Yes||4||No|
|3||Male||76||No||III||5||0||IO closure + deep curettage||Yes||eASC||Partial||0||No|
|4||Male||57||No||III||12||0||IO closure + deep curettage||Yes||eASC||Yes||0||Yes|
|5||Female||45||Yes||IV (multiple tracts)||6||12||IO closure + deep curettage||Yes||eASC||Partial||8||Yes|
|6||Male||35||Yes||II (stenosis)||5||Unknown||IO closure + deep curettage||No||eASC||No||0||No|
|7||Male||40||Yes||III||3||Unknown||IO closure + deep curettage||Yes||eASC||Yes||0||Yes|
|8||Male||59||No||II||11||13||Flap + deep curettage||No||eASC Allog||Partial||5||No|
|10||Female||28||No||III||5 + ileostomy||Not evaluable||Flap + partial fistulectomy||Yes||SVF||Yes||Not evaluable||Yes|
Autologous expanded adipose-derived stem cells (eASC) were selected in eight cases. Another case was treated using stromal vascular fraction (SVF) and in the last one, allogeneic adipose derived stem cells (Allo-eASC) were employed.
Both eASC (autologous and allogeneic) and SVF protocols were approved by the Ethics Committee of La Paz University Hospital in accordance with Spanish law, and by the Spanish Medical Agency according to European Medicine Agency (EMA) guidelines. All patients signed a detailed informed consent prior to any intervention, which included permission for data publication. Our institutional Committee on Human Experimentation (La Paz University Hospital) supervised all interventions performed. All ethical standards were in accord with those of the Helsinki Declaration (1975).
The liposuction was performed by a plastic surgeon and obtained 80-100 mL of fat. Phosphate buffered saline (PBS; Gibco of Thermo Fisher Scientific, Waltham, MA, United States) was used to wash the raw lipoaspirate and remove local anesthetics and cells. To extract the cellular fraction, the washed fat was digested with type I collagenase (Gibco) at a final concentration of 0.075% in saline solution at 37 °C for 45 min.
Collagenase was inactivated with Dulbecco’s modified Eagle’s medium (DMEM; Gibco) containing fetal bovine serum (10% v/v). Cells in suspension were then centrifuged for 10 min (250 ×g) and PBS was used again to wash the pellet. Centrifugation was repeated and afterwards the remaining erythrocytes were lysed by treating the suspension with ammonium chloride 160 mmol/L for 10 min at room temperature. To conclude the cellular extraction, a final wash and a filtration of the product through a 40 μm nylon mesh was performed.
Before injection, cells were suspended in sterile ringer-lactate solution (Griffols S.A., Barcelona, Spain). Morphologic determinations and phenotypic analyses were performed during product obtention. Data are partly published in García-Olmo et al. The cell viability was always > 95% as determined by trypan-blue (Sigma-Aldrich, St Louis, MO, United States).
The released cellular fraction (SVF) was seeded at 2-3 × 104 cells/cm2. Culture was carried out in DMEM with 10% fetal bovine serum and 1% ampicillin/streptomycin. No additional supplements were added. The atmospheric conditions were 37 °C with a 5% CO2 atmosphere.
Cells were re-plated once 80% confluency was confirmed; their prior detachment was performed by trypsinization (trypsin-EDTA; Gibco). This cycle was repeated up to three times until the required number of cells for implantation was obtained. Due to logistics and personal issues, in two cases the cells were then frozen for preservation.
Morphologic determinations and phenotypic analyses were performed by flow cytometry during expansion. Mycoplasma was detected using a Myco Alert Mycoplasma Detection Kit (Cambrex Corp., East Rutherford, NJ, United States). Data are partly published in García-Olmo et al.
At least one week before the surgical intervention was scheduled, expanded ASCs were prepared (washed with PBS, trypsinized, and centrifuged). Their viability was checked (> 95%), and finally the cells were resuspended in ringer-lactate solution (Griffols S.A.) at the desired volume and concentration (depending on the fistula) for their immediate use.
These cells were manufactured from donors by Tigenix SAU (Madrid, Spain) according to EMA permissions and regulations from healthy donors. The expansion protocol was similar to that of autologous procedures.
All surgical procedures were performed at La Paz University Hospital (Madrid), by the same team of surgeons, belonging to the Colorectal Surgery Unit.
In all cases, a deep curettage of the tracts was first performed, and then the ASC suspension (50%) was injected through a long, fine needle into the tract walls. The injections were superficial; not deeper than 2 mm. In seven cases, the fistulous tract was sealed with fibrin glue (Baxter Inc., Deerfield, IL, United States) containing a portion (1 mL) of the cells. The fibrin glue was used as a sealant to finalize the procedure in order to ensure cells remained in the fistulous area. The main reason for injecting a percentage of the ASC into the fibrin glue was to have a reservoir in the area so they could act for longer. However, recent investigations indicate that cells alone are sufficient for a therapeutic effect[17,18].
In very complex perianal fistulae, a partial fistulectomy was performed without removing intra-sphincteric tracts. The closure of the internal opening was achieved by stitches in six cases and by a mucosal advancement-flap in three cases. In the remaining patient, a fistulotomy was performed.
A first evaluation was performed on the 8th postoperative week, and a final evaluation was scheduled one year after the procedure (although patients attended the outpatient clinic in between, at variable intervals). Response was defined as a complete cessation of suppuration on week 8, despite not achieving a complete re-epithelization. Partial response was defined as an evident decrease in suppuration. Healing was defined as no suppuration from the external orifice, achieving a complete re-epithelization after one year of follow-up. These intervals of time for follow-up were selected following published data about the best periods for long-term fistula follow-up evaluation.
Of the ten highly recurrent perianal fistulae treated, 6/10 (60%) showed a clinical response 8 wk after the procedure, and 3/10 (30%) showed a partial response. One year later, 6/10 (60%) remained healed, with the external opening being completely epithelialized (Table 1). Postoperative results of Wexner Scores for Incontinence were 0 in 6 cases. In the two patients with previous fecal incontinence, the scoring improved from 12 to 8 and from 13 to 5. No adverse reactions or complications related to stem-cell therapy were reported during the study period.
No statistical relationships have been established between the use of fibrin glue, surgical approach or cell lineage, due to the small number and variability of patients.
Following strict regulations, we treated ten patients with recurrent perianal fistulae, achieving a 90% response and a complete healing after one year in six cases, with no associated incontinence. Moreover, in two cases, previous incontinence was reduced. It is important to remark that the performance of this therapeutic strategy does not produce injury to the anal sphincter, because intrasphincteric-tract resection is not required. Although this is not a randomized controlled trial, the results are similar to those already published[12-14].
ASCs enlarge the therapeutic arsenal for anal fistulae, and can be considered an interesting tool for the regeneration/repair of wounds or chronically damaged tissues. The specific mechanism of action of ASCs is still under study, but it has been widely demonstrated that these cells improve healing. Two different biologic effects are responsible for this healing effect: proliferation and differentiation on the one hand, and immune regulation and local suppression of inflammation on the other.
According to the EMA, ASC treatments in the EU should be administered only under clinical trials or other controlled conditions, such as Compassionate-use Programs. It is important to remark that all clinical uses of stem cells outside of these regulatory conditions are considered illegal. This is clearly stated in the law RD 1015/2009. In this way, in February 2011, the EMA published a report on stem cell-based medicinal products. It expressed concerns about the unregulated use of medicinal products containing ASCs.
In this context, one of the limitations of our study is the small number of patients included. We believed that a limited surgical treatment supported by ASC could be beneficial for these patients; but only those that did not meet the inclusion criteria or were scheduled in control groups of our clinical trials could be selected for the present study.
Various randomized controlled trials using ASCs for the treatment of anal fistulae have been conducted, and all of them show an excellent safety profile. Nevertheless, the actual efficacy is difficult to assess. To date, there are 11 published papers including data on stem cell-based treatment of anal fistulae (Table 2). The first one was published in 2003, and the last was published very recently, in 2013. Eight of these have been published by Spanish groups[11-14,17,21-23], two other papers come from South Korea[20,24], and one from Italy. The majority refer to ASC treatment of Crohn’s disease-related fistulae. The Italian study was the only one to select bone marrow as the ASC source for the treated fistula. The rest of the studies employed autologous or allogeneic cells from adipose tissue. In all studies, cells were expanded, and a wide range of doses applied. Except for the Italian study, all procedures included closure of the internal opening, and in all cases the cell injections were intra-lesional. Over three hundred patients have been enrolled in these studies and the most important result is the assurance of the excellent safety profile of stem cells: no serious cell-related adverse events were described. Regarding efficacy, results show very different profiles, but about 40%-60% of patients achieved healing (Table 3).
|Ref.||Year||Condition||Study design||Cell source||Cell quantity (dose)||Intervention model|
|García-Olmo et al||2003||Recto-vaginal fistula in Crohn’s disease||Case report||Autologous eASC||1 × 107||Single arm|
|García-Olmo et al||2005||Enterocutaneous, recto-vaginal, perianal fistula in Crohn’s disease||Phase I||Autologous eASC||1-3 × 107 re-suspended in fibrin glue||Single arm|
|García-Olmo et al||2009||Perianal fistula with or without Crohn’s disease||Phase II||Autologous eASC||Not specified||Two arms: fibrin glue, fibrin glue + eASC|
|García-Olmo et al||2010||Recto-vaginal fistula in Crohn’s disease||Case report||Allogenic eASC||Not specified||Single arm|
|Ciccocioppo et al||2011||Enterocutaneous and complex perianal fistula in Crohn's disease||Case report||Expanded autologous bone marrow||5 × 107||Single arm|
|Cho et al||2012||Perianal fistula in Crohn’s disease||Phase I||Autologous eASC||Not specified||Single arm: dose escalation study|
|Herreros et al||2012||Complex perianal fistula without Crohn’s disease||Phase III||Autologous eASC||2 × 107 then 4 × 107 if no effect||Three arms: fibrin glue, eASC, fibrin glue + eASC|
|Herreros et al||2012||Complex perianal fistula without Crohn’s disease||Observational||Autologous eASC||2 × 107 then 4 × 107 if no effect||Three arms: fibrin glue, eASC, fibrin glue + eASC|
|Guadalajara et al||2012||Perianal fistula with or without Crohn’s disease||Observational||Autologous eASC||Not specified||Two arms: fibrin glue, fibrin glue + eASC|
|de la Portilla et al||2012||Perianal fistula in Crohn’s disease||Phase I/II||Allogeneic eASC||2 × 107 then 4 × 107 if no effect||Single arm|
|Lee et al||2013||Perianal fistula in Crohn’s disease||Phase II||Autologous eASC||Depending on the fistula. Re-dosing (1.5 times) if no effect||Single arm|
|Ref.||Procedure||No. of patients treated||Healed (n)||Follow-up (mo)||Recurrence (n)||SAE (n)|
|García-Olmo et al||Closure of IO. Injection in site, without fibrin glue||1||1||3||0||0|
|García-Olmo et al||Cells resuspended in fibrin glue. Injection in site||9||6||12||Not specified||0|
|García-Olmo et al||Closure of IO. Injection in site||Fibrin glue: 25 Fibrin glue + eASC: 24||Fibrin glue: 3 Fibrin glue + eASC: 17||12||Fibrin glue: 0 Fibrin glue + eASC: 2||4 (1 related to fibrin glue, others unrelated)|
|García-Olmo et al||Closure of IO. Injection in site, without fibrin glue||1||1||36||1||0|
|Ciccocioppo et al||Four injections in site||10||7||12||0||0|
|Cho et al||Closure of IO and fibrin glue. Injection in site||9||3||15||0||0|
|Herreros et al||Closure of IO. Injection in site||eASC: 64 Fibrin glue + eASC: 60 Fibrin glue: 59||eASC: 27 Fibrin glue + eASC: 24 Fibrin glue: 23||6||eASC: 0 Fibrin glue + eASC: 4 Fibrin glue: 0||4 unrelated to study treatment|
|Herreros et al||Closure of IO. Injection in site||Not specified||eASC: 57% Fibrin glue+ eASC: 52.4% Fibrin glue: 37.3%||12||Not specified||1 unrelated to study treatment|
|Guadalajara et al||Closure of IO. Injection in site||Fibrin glue: 13 Fibrin glue + eASC: 21||Fibrin glue: 3 Fibrin glue + eASC: 10||38||Fibrin glue: 1 Fibrin glue + eASC: 5||0|
|de la Portilla et al||Closure of IO. Injection in site, without fibrin glue||24||9||4||Not specified||2 unrelated to study treatment|
|Lee et al||Injection in site and fibrin glue||43||27||12||4||0|
The cost of the treatment is another important issue. Nowadays, the production cost of expanded ASCs (Good Manufacturing Practice compliant) can range from € 8000-12000, though the production could be reduced to € 3000-4000 without expansion. We estimate that a high scale industrial production could significantly reduce the expenses. A reasonable strategy that we propose, considering the high cost of cell expansion and our previous results, would be to apply a first treatment with SVF, freeze a portion of the cells obtained, and propose a second treatment with expanded cells in cases achieving no response after eight weeks.
Treatment of recurrent fistulae is a difficult surgical challenge. In these patients, the pursuit of healing usually involves multiple operations, with a subsequent perianal scarring and distortion. In the most complex cases, the condition is worsened by the accompanying fecal incontinence. Therefore, these individuals are progressively more difficult to treat, resulting in the exasperation of both the patient and the surgeon. In these cases, wound healing is a critical issue, and indeed, new approaches are needed. For the reasons outlined earlier, we believe that once available, ASCs will fulfill a clear and previously unmet medical need, helping to improve the healing and hence the quality of life of patients with recurrent perianal fistulae.
In conclusion, limited surgery supported by ASCs may constitute as a new therapeutic strategy in the treatment of recurrent fistulae.
The authors thank Carolina Gonzalez and Tihomir Georgiev for their collaboration in all our Adipose Stem Cells Trials and Studies, assisting in different technical aspects and taking care of the patients.
The concept of stem cell therapy came from the possibility of obtaining an immature cell that could differentiate into a specific lineage if placed in the correct environment. One of the first examples was the transplantation of stem cells in hematologic patients, which could achieve the regeneration of normal marrow, and is now widely used. Following this idea, researchers raised the question of whether or not there could be different stem cells for other organs, or even embryonic cells that could develop into any of them. The study and therapeutic use of these types of cells could be the answer for many incurable injuries and diseases.
Complex and recurrent anal fistulae (whether associated or not to Crohn’s disease), constitute an important surgical problem, which can be difficult to solve. Many strategies have been proposed to achieve healing, including different surgical techniques, including fibrin glues and plugs. The application of stem cells in the fistula tract promotes the “closure” of the fistula by stimulating the regeneration of the tissue, both by direct growth and immunomodulatory effects. The exact mechanisms by which stem cells induce healing are still under investigation.
An increasing number of randomized controlled trials have tested the application of adipose-derived stem cells (ASCs) in perianal fistulae, with variable rates of success. In the present study, we applied ASCs to the worst patients, those with recurrent fistulae despite multiple previous treatments and interventions. In these desperate cases, even a partial response to the treatment was a success, as patients’ distress was a constant after so many failures. ACSs were remarkably effective and we achieved healing in 60% after one year of follow-up.
This study, and various others in a randomized controlled trial setting, suggests that the surgical application of stem cells in anal fistula tract is a potentially therapeutic strategy that could resolve even the most recurrent and complex fistulae. In the same direction, ACSs are being used for the regeneration of skin, cartilage, bone, cornea, endothelium, etc. Applications to other organs, such as the heart and lung, and the nervous system have raised high expectations in the field. Research continues, and new applications are sure to develop in the near future.
An anal fistula is an abnormal conduct communicating the anal canal with the perianal skin. The fistula tract typically breaks through the sphincters, and can have multiple ramifications. If one of the openings is blocked, an abscess occurs, which can worsen the condition. Drainage of these abscesses and surgical attempts to close the fistula can damage the muscle of the sphincters and cause fecal incontinence. The former, associated to suppuration and pain are the most common symptoms, creating a permanent discomfort for patients. Adult (somatic) stem cells are undifferentiated cells that can be found in differentiated tissue (such as bone, fat, and muscle) and have the potential to give rise to the specialized cell types present in the tissue from which they originate. A stem cell fulfills three characteristics: self-renewal capacity, differentiation potential, and in vivo engraftment capacity. Stem cell therapy: use of stem cells to replace those from damaged or diseased tissue. The source of the cells can either be the patient (autologous), another individual (allogeneic), or an animal (xenogeneic).
A good and interesting study even though it includes only ten patients. However, the results are very useful to speculate about the best current treatment of recurrent complex fistulae. It will be interesting if a randomized cross-over multicenter study can confirm these results with stem cell therapy in the complex anal fistulae.
|1.||García-Aguilar J, Davey CS, Le CT, Lowry AC, Rothenberger DA. Patient satisfaction after surgical treatment for fistula-in-ano. Dis Colon Rectum. 2000;43:1206-1212. [PubMed]|
|2.||Whiteford MH, Kilkenny J, Hyman N, Buie WD, Cohen J, Orsay C, Dunn G, Perry WB, Ellis CN, Rakinic J. Practice parameters for the treatment of perianal abscess and fistula-in-ano (revised). Dis Colon Rectum. 2005;48:1337-1342. [PubMed] [DOI]|
|3.||Ortíz H, Marzo J. Endorectal flap advancement repair and fistulectomy for high trans-sphincteric and suprasphincteric fistulas. Br J Surg. 2000;87:1680-1683. [PubMed] [DOI]|
|4.||Garcia-Olmo D, Garcia-Arranz M, Herreros D. Expanded adipose-derived stem cells for the treatment of complex perianal fistula including Crohn’s disease. Expert Opin Biol Ther. 2008;8:1417-1423. [PubMed] [DOI]|
|5.||Trebol Lopez J, Georgiev Hristov T, García-Arranz M, García-Olmo D. Stem cell therapy for digestive tract diseases: current state and future perspectives. Stem Cells Dev. 2011;20:1113-1129. [PubMed] [DOI]|
|6.||García-Gómez I, Elvira G, Zapata AG, Lamana ML, Ramírez M, Castro JG, Arranz MG, Vicente A, Bueren J, García-Olmo D. Mesenchymal stem cells: biological properties and clinical applications. Expert Opin Biol Ther. 2010;10:1453-1468. [PubMed] [DOI]|
|7.||Falanga V, Iwamoto S, Chartier M, Yufit T, Butmarc J, Kouttab N, Shrayer D, Carson P. Autologous bone marrow-derived cultured mesenchymal stem cells delivered in a fibrin spray accelerate healing in murine and human cutaneous wounds. Tissue Eng. 2007;13:1299-1312. [PubMed] [DOI]|
|8.||Wu Y, Chen L, Scott PG, Tredget EE. Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis. Stem Cells. 2007;25:2648-2659. [PubMed] [DOI]|
|9.||Chen L, Tredget EE, Wu PY, Wu Y. Paracrine factors of mesenchymal stem cells recruit macrophages and endothelial lineage cells and enhance wound healing. PLoS One. 2008;3:e1886. [PubMed] [DOI]|
|10.||Chaparro M, Zanotti C, Burgueño P, Vera I, Bermejo F, Marín-Jiménez I, Yela C, López P, Martín MD, Taxonera C. Health care costs of complex perianal fistula in Crohn’s disease. Dig Dis Sci. 2013;58:3400-3406. [PubMed] [DOI]|
|11.||García-Olmo D, García-Arranz M, García LG, Cuellar ES, Blanco IF, Prianes LA, Montes JA, Pinto FL, Marcos DH, García-Sancho L. Autologous stem cell transplantation for treatment of rectovaginal fistula in perianal Crohn’s disease: a new cell-based therapy. Int J Colorectal Dis. 2003;18:451-454. [PubMed] [DOI]|
|12.||García-Olmo D, García-Arranz M, Herreros D, Pascual I, Peiro C, Rodríguez-Montes JA. A phase I clinical trial of the treatment of Crohn’s fistula by adipose mesenchymal stem cell transplantation. Dis Colon Rectum. 2005;48:1416-1423. [PubMed] [DOI]|
|13.||Garcia-Olmo D, Herreros D, Pascual I, Pascual JA, Del-Valle E, Zorrilla J, De-La-Quintana P, Garcia-Arranz M, Pascual M. Expanded adipose-derived stem cells for the treatment of complex perianal fistula: a phase II clinical trial. Dis Colon Rectum. 2009;52:79-86. [PubMed] [DOI]|
|14.||Herreros MD, Garcia-Arranz M, Guadalajara H, De-La-Quintana P, Garcia-Olmo D. Autologous expanded adipose-derived stem cells for the treatment of complex cryptoglandular perianal fistulas: a phase III randomized clinical trial (FATT 1: fistula Advanced Therapy Trial 1) and long-term evaluation. Dis Colon Rectum. 2012;55:762-772. [PubMed] [DOI]|
|15.||Parks AG, Gordon PH, Hardcastle JD. A classification of fistula-in-ano. Br J Surg. 1976;63:1-12. [PubMed]|
|16.||Vaizey CJ, Carapeti E, Cahill JA, Kamm MA. Prospective comparison of faecal incontinence grading systems. Gut. 1999;44:77-80. [PubMed]|
|17.||de la Portilla F, Alba F, García-Olmo D, Herrerías JM, González FX, Galindo A. Expanded allogeneic adipose-derived stem cells (eASCs) for the treatment of complex perianal fistula in Crohn’s disease: results from a multicenter phase I/IIa clinical trial. Int J Colorectal Dis. 2013;28:313-323. [PubMed] [DOI]|
|18.||Singer NG, Caplan AI. Mesenchymal stem cells: mechanisms of inflammation. Annu Rev Pathol. 2011;6:457-478. [PubMed] [DOI]|
|19.||Ortiz H, Marzo M, de Miguel M, Ciga MA, Oteiza F, Armendariz P. Length of follow-up after fistulotomy and fistulectomy associated with endorectal advancement flap repair for fistula in ano. Br J Surg. 2008;95:484-487. [PubMed] [DOI]|
|20.||Lee WY, Park KJ, Cho YB, Yoon SN, Song KH, Kim do S, Jung SH, Kim M, Yoo HW, Kim I. Autologous adipose tissue-derived stem cells treatment demonstrated favorable and sustainable therapeutic effect for Crohn’s fistula. Stem Cells. 2013;31:2575-2581. [PubMed] [DOI]|
|21.||Garcia-Olmo D, Herreros D, Pascual M, Pascual I, De-La-Quintana P, Trebol J, Garcia-Arranz M. Treatment of enterocutaneous fistula in Crohn’s Disease with adipose-derived stem cells: a comparison of protocols with and without cell expansion. Int J Colorectal Dis. 2009;24:27-30. [PubMed] [DOI]|
|22.||García-Olmo D, Herreros D, De-La-Quintana P, Guadalajara H, Trébol J, Georgiev-Hristov T, García-Arranz M. Adipose-derived stem cells in Crohn’s rectovaginal fistula. Case Rep Med. 2010;2010:961758. [PubMed] [DOI]|
|23.||Guadalajara H, Herreros D, De-La-Quintana P, Trebol J, Garcia-Arranz M, Garcia-Olmo D. Long-term follow-up of patients undergoing adipose-derived adult stem cell administration to treat complex perianal fistulas. Int J Colorectal Dis. 2012;27:595-600. [PubMed] [DOI]|
|24.||Cho YB, Lee WY, Park KJ, Kim M, Yoo HW, Yu CS. Autologous adipose tissue-derived stem cells for the treatment of Crohn’s fistula: a phase I clinical study. Cell Transplant. 2013;22:279-285. [PubMed] [DOI]|
|25.||Ciccocioppo R, Bernardo ME, Sgarella A, Maccario R, Avanzini MA, Ubezio C, Minelli A, Alvisi C, Vanoli A, Calliada F. Autologous bone marrow-derived mesenchymal stromal cells in the treatment of fistulising Crohn’s disease. Gut. 2011;60:788-798. [PubMed] [DOI]|