Research articles on stem cell effectiveness for Knees Hips & Shoulders

A prospective multi-site registry study of a specific protocol of autologous bone marrow concentrate for the treatment of shoulder rotator cuff tears and osteoarthritis

Abstract

Introduction Shoulder pain is a common musculoskeletal complaint in the general population. Bone marrow concentrate (BMC) injections offer promising potential as a minimally invasive approach for treatment of shoulder pain in degenerative disease. In this study, we investigated the clinical outcomes of the BMC injections for treatment of shoulder pain and disability due to osteoarthritis (OA) and rotator cuff tears in a treatment registry population.

Methods A total of 115 shoulders in 102 patients were treated with autologous BMC injections for symptomatic OA at the glenohumeral joint and/or rotator cuff tears. Data were collected for factors potentially influencing outcome, including age, sex, body mass index, and the type of condition treated (ie, OA or rotator cuff tear). Clinical outcomes were assessed serially over time using the disabilities of the arm, shoulder and hand score (DASH), the numeric pain scale (NPS), and a subjective improvement rating scale. Baseline scores were compared to the most recent outcome scores at the time of the analysis and adjusted for demographic differences. We reported comparisons of pre- and post-treatment scores, the differences between osteoarthritis and rotator cuff groups, and the predictive effects on the clinical outcomes.

Results At the most current follow-up assessment after treatment, the average DASH score decreased (improved) from 36.1 to 17.1 (P<0.001) and the average numeric pain scale value decreased (improved) from 4.3 to 2.4 (P<0.001). These changes were associated with an average subjective improvement of 48.8%. No differences were observed between outcomes among the shoulders treated for OA versus rotator cuff tears, nor did age, sex, or body mass index influence pain or functional outcomes. There were no significant treatment-related adverse events reported.

Discussion We observed preliminarily encouraging results following BMC injections for shoulder OA and rotator cuff tears. These results serve as basis for the design of an adequately powered randomized controlled trial.

Introduction

Shoulder pain is the third most common musculoskeletal disorder observed in the primary care setting, after back and neck pain.1,2 Estimates of the point prevalence of shoulder pain in adults range from 7% to 27% in the population under the age of 70 and from 13% to 26% in adults 70 years and over.3 The lifetime prevalence of painful shoulder disorders is 10% in the United States, with an annual incidence of 15 new cases per 1,000 in the at-risk population.4 The insidious (ie, non-traumatic) onset of shoulder pain is attributed to various degenerative and inflammatory processes, including disorders of the rotator cuff, adhesive capsulitis, and glenohumeral osteoarthritis (OA).5 In patients >70 years, the most common diagnosis associated with shoulder pain is a rotator cuff derangement.6 Persistent inflammatory and degenerative conditions are responsible for recurrent or chronic shoulder pain in 40% of patients, and disability associated with chronic shoulder pain significantly impacts the economy in the form of decreased productivity and health care costs.

Arthroscopic surgery is a common approach for treating shoulder pain, and from 1996 to 2006, the number of these procedures increased by 600%, including an overall 115% increase in the number of rotator cuff repairs.9 Arthroscopic surgery is technically challenging and complications or residual impairment related to the procedure, including stiffness, implant failure, nerve injury, and adhesive capsulitis, are estimated to range between 5.8% and 9.5%.10,11 It is estimated that recurrent defects occur in a very high proportion of cases, post-surgically.12,13

As an alternative to surgery, cell-based regenerative therapies, including the use of mesenchymal stem cells (MSCs), have shown promising results for the treatment of degenerative conditions of joints.14 MSCs are multipotent stem cells with the ability to differentiate into bone, cartilage, adipose, and muscle cells, and thus provide a means of facilitated tissue repair.15 Bone marrow is a rich source of MSCs, with the isolation and autologous transplantation of MSCs from bone marrow concentrate (BMC) having the advantage of avoiding immunogenic complications potentially associated with the use of allogeneic cell transplants.15,16

The clinical use of MSCs as an adjunct to surgical treatment of shoulder disorders has been described previously in the literature,17,18 including a study of 90 rotator cuff arthroscopy cases with a reported 100% positive outcome rate for procedures that used MSC-enriched BMC as an adjunctive therapy to the surgeries, a substantial increase in positive outcomes in comparison with procedures that did not use MSCs.19

Currently, there are no clinical studies that describe the treatment of painful shoulder conditions with BMC and MSCs alone. The clinical use of BMC injections for treatment of shoulder diseases requires analysis of the effectiveness of this approach. In the present study, we report on the symptomatic and functional outcomes for patients with painful shoulder disorders who were treated with BMC injections. As part of the analysis, we examined the impact of age, sex, and body mass index (BMI) on the reported outcomes.

Methods

Study participants and data collection Patients were selected from a treatment registry designed to track the safety and efficacy of patients presenting to a network of 13 clinics providing treatment of joint disorders using autologous stem cells, BMC, or platelet-rich plasma (PRP). The registry was designed as an ongoing prospective survey system, using an automated questionnaire generated at 1 month, 3 months, 6 months, and 12 months and then annually post treatment, via an electronic database system using ClinCapture software (Clinovo Clinical Data Solutions, Sunnyvale, CA, USA; https://www.clinovo.com/clincapture). Baseline data are entered by registry staff who are also tasked with telephonic follow-up of patients who fail to respond to the electronic survey.

In the present study, patients with presenting symptoms of shoulder pain who were subsequently diagnosed with glenohumeral OA and/or partial or full-thickness rotator cuff tears were culled from the registry. Shoulder pathology was assessed by magnetic resonance imaging (MRI) and supported by findings from physical examination. All the patients included in the study had failed conservative therapies, such as physical therapy, medications, and test of time. Patients with less than a 3-month follow-up or a rotator cuff tear greater than 1.5 cm and evidence of retraction were excluded.

Procedure description Patients were restricted from using corticosteroids and non-steroidal anti-inflammatory drugs (NSAIDs) for 2 weeks preceding the procedure. This restriction was placed based on in vitro research revealing that corticosteroids and NSAIDs can have an inhibitory effect on MSCs.20 To prompt a brief inflammatory response before receiving the BMC, patients were pre-injected with a hypertonic dextrose solution into the joint structures.21 Ultrasound or fluoroscopy was used to guide intra-articular or rotator cuff tear needle placement. When fluoroscopy was used to confirm intra-articular needle placement, iodixanol (Visipaque, NDC# 0407-2223-06) radiographic contrast agent was injected followed by a second injection of 3–5 mL of 12.5% dextrose (NDC# 0409-6648-02) and 0.1% lidocaine (NDC# 0409-4276-02) or 0.25% ropivicaine (NDC# 63323-286-23) in normal saline (NDC# 0409-4888-50). Two to five days after the pre-injection, again using ultrasound or fluoroscopic guidance, 10–15 mL of bone marrow aspirate per bone site was collected from the patients’ posterior superior iliac crest (total 60–90 mL) into heparinized syringes. For each 1 mL of whole bone marrow aspirate collected, 1,000 units of heparin (NDC# 25021-403-01 and 25021-404-01) was added and the cell suspension was serially centrifuged, following which 1–3 mL of coagulated plasma containing white blood cells (termed buffy coat or the middle layer of the centrifuged bone marrow aspirate) was collected by manual serological pipetting. In addition to BMC isolation, 60 mL of intravenous blood was drawn for the isolation of PRP and platelet lysate (PL). These platelet products were used due to numerous studies demonstrating that the growth factors they contain can cause MSC proliferation.22 PRP was prepared by low-speed centrifugation (200× g) to separate plasma and buffy coat layers from red blood cells and stored at −20°C. Subsequent PL was isolated by recentrifugation of PRP. All cell preparations were performed in sterile conditions under an ISO-5 class laminar flow cabinet located in an ISO-7 class clean room. The BMC injectate, containing PRP and PL, was transported via sterile means back to the operating room, where it was injected into the intra-articular structure and/or the rotator cuff tear under fluoroscopy or ultrasound guidance.

Predictive factors Predictive factors examined in the study were age, BMI, sex, and type of the joint disease (ie, OA or rotator cuff tear). Rotator cuff tears were defined as including damage to the supraspinatus, infraspinatus, subscapularis, teres major, or teres minor tendons/muscles. Joints that had rotator cuff disorder associated with OA were classified in the rotator cuff group as the predominant cause of symptoms.

Cell count data Cell count represents the total number of nucleated cells aspirated from the bone marrow and injected into the shoulder joint. Cell count data were accessed from a laboratory database (Centeno Schultz Clinic, Broomfield, CO, USA). Data were available for only one clinic. For cell counting, 5 μL samples were obtained and red blood cells were lysed in 995 μL of sterile distilled water (Thermo Fisher Scientific, Waltham, MA, USA). The number of cells was then manually counted for four times under a microscope (National Optical, Schertz, TX, USA) using a hemocytometer (Reichert Bright-Line; Hausser Scientific, Horsham, PA, USA). The average of the four counts was calculated. The cell count was obtained by multiplying the dilution factor, volume of the hemocytometer, and final volume of the sample.

Outcomes The outcomes of interest were the disabilities of the arm, shoulder and hand (DASH) numeric pain scale (NPS) and the subjective improvement rating score as reported by the patient. The DASH is a validated functional scale that measures the disability of shoulder and upper extremities.23 The DASH score is derived from answers to 30 questions assessing various aspects of daily and recreational activities, in addition to specific symptoms, including pain, tingling, stiffness, and weakness;24 and ranges from 0 (no disability) to 100 (most severe disability).23

Pain severity was assessed using the NPS, a one-item questionnaire with eleven scoring levels ranging from 0 (no pain) to 10 (most severe pain). The NPS scale is a valid means of assessing various types of pain.25,26 The minimal clinically important difference (MCID) was defined by 2-point reduction in the NPS score and 10-point reduction in the DASH score.23,26

For the subjective percentage improvement rating metric, patients were asked: “Compared to your condition prior to the procedure, what percent difference have you seen in your condition?” The response range was from −100% (significantly worsened) to 100% (significantly improved), with zero indicating no change.

For each outcome metric, the baseline score was compared with the most recent score of 3 months or more months duration post treatment.

In order to track adverse events, patients were asked the following questions: “Did you experience any complications you believe may be due to the procedure (ie, infection, illness, etc)? If yes, please explain” and “Have you been diagnosed with any new illness since the procedure? If yes, please explain.”

Statistical analyses Pre-treatment (baseline) and most recent post-treatment clinical scores were reported using the means and standard deviations. The differences between the two scores were then examined using the Wilcoxon signed-rank test for dependent groups. The outcome differences (changes in the clinical scores) between OA and rotator cuff disorders were analyzed using the Wilcoxon rank sum test. Outcome analysis was performed on the most recent post-treatment clinical score, using the method of last observations carried forward. Analysis of outcome scores at each time-point was not plausible in the current study due to the magnitude of missing data.

We also examined the frequency and proportion of joints that achieved the MCID (responders to treatment) and joints that failed to achieve the MCID for the DASH and NPS scales (non-responders to treatment). Wilcoxon rank sum and Fisher exact tests were applied to test the demographical differences (age, BMI, and sex) between responders and non-responders.

Linear regression analysis was used to examine the effect of predictive variables on the symptomatic and functional outcomes. First, models were constructed to examine the effect of baseline score, disease type, age, BMI, and sex on outcomes. Second, models were also constructed to examine the effect of cell count in addition to other covariates.

Responding to questionnaires (follow-up surveys) was analyzed. Responder bias was assessed by examining differences between baseline values for the non-responders versus the responders using logistic regression analysis. The analysis was adjusted for age, sex, and BMI.

SAS software version 9.4 was used for all analyses.27 The post hoc power analysis described in the discussion was performed using G*Power 3.1 software.28

Results During the period of September 2010 through January 2014, there were 115 shoulder joints in 102 patients meeting the inclusion criteria who were treated with same-day BMC injection procedures. Among the 115 shoulders, there were 81 (70.4%) diagnosed with a rotator cuff tear and 34 (29.6%) diagnosed with OA alone. See Table 1 for the demographic characteristics of the groups. DASH scores decreased by an average of 52.6%, from 36.1 at baseline to 17.1 at final follow-up (P<0.001). NPS decreased by 44.2%, from 4.3 to 3.4 (P<0.001). The average self-rated improvement was 48.8% compared to baseline (Table 2). The reduction in disability and pain was observed starting at the first month post treatment; mean DASH and NPS scores were 18.5 and 2.6, respectively (number of observations [Ns] =25 for DASH; 30 for NPS). Improvement continued up to 2 years after treatment with means of 3.3 and 1.5 for DASH and NPS scales, respectively (Ns =3 for DASH; 8 for NPS).

The follow-up scores (last observations carried forward) for DASH and NPS were available for 40 and 55 joints, respectively. A total of 32 joints (58.2%) achieved the MCID on the NPS scale (2 points reduction) and 26 joints (65%) achieved the MCID on the DASH scale (10 points reduction). Demographical analysis showed that responders to treatment on the DASH scale (ie, joints achieving the MCID) were younger (mean age =56.1 years) and had higher female proportion (46.2%); non-responders (joints failing to achieve the MCID) were 7.1% female and 65.3 years old in average (P-values =0.025 and 0.015 for age and sex comparisons, respectively). Regarding the NPS scale, there were no demographical differences between responders to treatment and non-responders.

There were no differences in outcomes between the OA and rotator cuff groups in either the univariate (Table 3) or multivariate analysis (Table 4). Age, BMI, and disease type did not demonstrate a measurable effect on the functional (DASH) and pain (NPS) outcomes in the linear regression model (Table 4). Although males reported a lower subjective improvement rating score, the effect of sex was not significant on DASH and NPS outcomes. DASH and NPS changes were significantly associated with the respective baseline scores (P-value <0.001).

Discussion

Shoulder patients treated with BMC injections demonstrated substantial symptomatic and functional improvement at follow-up. Functional improvements exceeded the minimally important difference defined as a 10-point change on the DASH scale.23 The 44.2% reduction in pain also exceeded the minimum important difference as defined by 30% decrease on the NPS scale.26 Improvement in pain and disability was observed at first month post-treatment and continued up to 2 years, which was the latest time-point obtained for the DASH and NPS scales. No serious adverse events were reported after the procedures.

Bone marrow aspirate is a rich source of MSCs, and the treatment effects observed in this study may be related to the regenerative characteristics of these cells.29 The regenerative capacity of bone marrow-derived MSCs has been demonstrated in several animal studies.30,31 These studies have demonstrated that MSC transplantation both repairs damaged tissue and restores function. The use of BMC for the treatment of shoulder conditions has already partly translated to clinical use; in one study of BMC use with rotator cuff surgery, the authors reported reduced retear rates, an indication of improved tendon integrity.19 The results in the present study are consistent with these results, but the more invasive surgery is absent.

Although age and sex were significantly different between responders to treatment and non-responders (as measured by DASH), the adjusted multivariate analyses showed that age, sex, and BMI did not predict the functional and pain outcomes. Our finding that neither age nor BMI had an impact on outcomes was also consistent with the findings of prior authors reporting on rotator cuff repairs and joint arthroplasty.32,33Our finding that women reported greater subjective improvement ratings compared to males was surprising given that the functional and pain scales did not indicate better outcomes for women. This latter finding is also consistent with the reports from studies of shoulder surgery, in which sex has not been found to predict pain or functional outcome.34,35 Two recent studies have shown that joint’s functional outcomes can be affected by sex, but it is unclear whether the sex effect is due to biological and/or anatomical differences between males and females or due to confounding factors.36,37 Patients with higher disability and pain metrics at baseline experienced the greatest improvements, possibly just an indication of regression to the mean, rather than an indication that the procedure is more successful in patients with more substantial symptoms. The finding that rotator cuff involvement did not affect outcome was consistent with previous reports of the efficacy of biologics and cell-based therapies for such conditions.19,38

The effect of stem cell dose variation on the efficacy of musculoskeletal injections is still unclear, and prior reports have been inconsistent.39–41 This is probably due to the fact that different methods, sources, cell doses, and types were used for the treatment of orthopedic disease. In this study, the number of nucleated cells within the BMC injection did not significantly impact clinical outcomes. Although we did not find a significant association between the injected cell dose and treatment outcome in this study; we emphasize that these are the results of a small group, and larger population studies are required to further explore the role of cell dose in the treatment of shoulder disease.

Limitations of our study include the subjective nature of the orthopedic scales and the inability to discriminate between subclasses of pathology in the registry data, as the dichotomous classification of either joint OA or rotator cuff does not capture the nuanced differences in disease severity in the group, and thus, there is no way to know why some patients responded better than others.

As a treatment registry study, there was a lack of a control group, which is always a concern with outcome studies. This is less of a concern for the size or even the validity of the effect observed, as the patients all served as their own controls. A larger issue is the fact that a placebo or other effect not related to the cellular/biologic therapy cannot be ruled out as the cause of the observed outcomes, nor can the effect of the BMC be differentiated from the PRP or hypertonic dextrose. As the final outcome values were recorded, an average of 7–11 months after the treatment, a placebo response seems unlikely.

Although we used both PRP and hypertonic dextrose in conjunction with the BMC injections, neither were employed for their curative effects on their own, nor was there reason to believe that either PRP or hypertonic dextrose would have a significant impact on the conditions treated in this study. Clinical studies of the efficacy of PRP injections alone for rotator cuff tears have not demonstrated significant benefit for the therapy.42,43 Although there are no studies of PRP for glenohumeral OA, studies of the therapy for the knee have shown either minimal or temporary benefit.44 Likewise, we were unable to find any publications, including animal models, indicating that dextrose alone can produce a healing effect of muscular or tendon injuries.45

There is additional concern regarding missing data and the possibility of the non-response bias. In the multivariate analyses, younger age and higher BMI were associated with non-response to the subjective improvement rating questionnaire, although non-response was not related to the severity of condition determined by baseline scores.

Despite these limitations, these study results are encouraging and portend what are potentially significant clinical implications. If indeed BMC injections can act as an intermediary treatment between non-invasive conservative care and rotator cuff surgery or joint arthroplasty, there is a large population of patients who could benefit. Our findings warrant further investigation with randomized and placebo-controlled studies, with long-term follow-up.

Conclusion

The use of BMC to treat symptomatic rotator cuff tears and glenohumeral OA is promising, and in an uncontrolled treatment registry population, effective at both reducing pain and improving function. Randomized clinical trials are required to confirm the efficacy of BMC injections for treatment of shoulder OA and rotator cuff tears.

Footnotes

Disclosure Dr Christopher Centeno is a shareholder and director of Regenerative Sciences, LLC. Hasan Al-Sayegh is an employee of the Centeno Schultz Clinic, Regenerative Sciences, LLC. Dr Jamil Bashir is a fellow trainer at the Centeno Schultz Clinic. Dr Shaun Goodyear and Dr Michael Freeman have no conflicts of interest.

References

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Direct transplantation of mesenchymal stem cells into the knee joints of Hartley strain guinea pigs with spontaneous osteoarthritis

Mitsuhiko Sato,1 Kenzo Uchida, 1 Hideaki Nakajima,1 Tsuyoshi Miyazaki,1 Alexander Rodriguez Guerrero,1 Shuji Watanabe,1 Sally Roberts,2 and Hisatoshi Baba1

Abstract

Introduction Mesenchymal stem cells (MSCs) can differentiate into various connective tissue cells. Several techniques have been used for the clinical application of MSCs in articular cartilage repair; however, there are many issues associated with the selection of the scaffold material, including its ability to support cell viability and differentiation and its retention and degradation in situ. The application of MSCs via a scaffold also requires a technically demanding surgical procedure. The aim of this study was to test the outcome of intra-articular transplantation of mesenchymal stem cells suspended in hyaluronic acid (HA) in the knee joints of Hartley strain guinea pigs with spontaneous osteoarthritis (OA).

Methods Commercially available human MSCs were cultured, labeled with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), suspended in either PBS or HA, and injected into the knee joints of 7-month-old animals. The control animals were injected with either PBS or HA alone. The animals were sacrificed at 1, 3, and 5 weeks post transplantation, the knee joints harvested, and fluorescent microscopic analysis was performed. Histological and immunohistochemical analysis were performed at 5 weeks post transplantation.

Results At 5 weeks post transplantation, partial cartilage repair was noted in the HA-MSC group but not in the other groups. Examination of CFDA-SE-labeled cells demonstrated migration, differentiation, and proliferation of MSC in the HA-MSC group. There was strong immunostaining for type II collagen around both residual chondrocytes and transplanted MSCs in the OA cartilage.

Conclusion This scaffold-free and technically undemanding technique appears to result in the regeneration of articular cartilage in the spontaneous OA animal model. Although further examination of the long-term effects of transplantation is necessary, the findings suggest that intra-articular injection of HA-MSC mixture is potentially beneficial for OA.

Introduction

Osteoarthritis (OA) of the knee joint is characterized pathologically by degeneration of articular cartilage, sclerosis of the subchondral bone, and marginal osteophyte formation, and is characterized clinically by chronic devastating pain and disability in the elderly. OA is a major public health problem and its prevalence is expected to increase dramatically and rapidly over the next 20 years with an increasingly aged population [1]. Although tibial osteotomy and total knee arthroplasty have been pursued in a large number of patients to eliminate joint pain and improve joint function, the majority of patients with knee OA are managed conservatively with medication and/or physiotherapy. Development of less technically demanding but effective therapies for knee OA, such as cell transplantation with or without scaffold enhancement, is therefore desirable.

Mesenchymal stem cells (MSCs) have the capacity to differentiate into a variety of connective tissue cells [2-6]. Several techniques have been used for the clinical application of MSCs in articular cartilage repair [7,8]. In general, the cells are delivered into either the cartilage or bone using a three-dimensional scaffold fixed to the articular defect site. There are many issues associated with the selection of the scaffold material, however, including its ability to support cell viability and differentiation and its retention and degradation in situ. Moreover, the application of MSCs via a scaffold usually requires a technically demanding surgical procedure. On the other hand, direct intra-articular injection of MSCs has only been carried out in a limited experimental setting [9,10]. In these animal studies, autologous MSCs - mixed with a dilute solution of sodium hyaluronan (hyaluronic acid (HA)) as a cell binding or cytotactic factor - were directly injected into the knee joint of surgically induced knee OA or focal cartilage defect in certain animal models. The procedure resulted in retardation of the progression of destruction of the degenerative cartilage. Although the injection of MSCs in HA may be the simplest approach clinically, disease progression is rapid in these models, thus making it less amenable to therapeutic intervention [11]. The potential outcomes of this method as a treatment for the slowly progressive process of cartilage degeneration, as commonly occurs in human OA, are still unknown.

The objective of the present study was to determine whether intra-articular injection of MSCs suspended in HA solution into the knee joint enhances the repair of degenerated cartilage in an animal model of spontaneous OA. We used Hartley strain guinea pigs because these animals spontaneously develop degenerative cartilage changes in the knee joint that mimic those of human OA [11-13]. The disease is generally bilaterally symmetrical on the medial tibial plateau in an area unprotected by the meniscus, and the earliest changes can be seen when animals are approximately 3 months old [12,13]. This animal model is more suitable for the evaluation of disease-modifying treatments.

Materials and methods

Experimental animals Seven-month-old male Hartley strain guinea pigs (n = 60) weighing 928 ± 4.8 g (mean ± standard deviation) were used. The experimental protocol met the Animal Care Ethics Committee Guidelines for Experimental Studies of Fukui Medical University and Institutional Review Board Guidelines for Stem Cell Research.

Preparation of human MSCs for in vivo tracing Human MSCs were purchased from Lonza (lot number PT-2501; Lonza, Walkersville, MD, USA). The cells were cultured at 37°C in a humidified atmosphere of 5% CO2 in mesenchymal stem cell growth medium (MSCGM™; Lonza). The medium was replaced every 3 days. Seven to 10 days after incubation, the cells reached 75% confluence in the seeded flask and were subcultured. Cells from passages three to five were used for the experiments. For in vivo tracing, the MSCs were labeled with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) (Vybrant CFDA SE Cell Tracer Kit (V-12883); Molecular Probes, Eugene, OR, USA) as described previously [14].

Intra-articular injection of MSCs The labeled cells were carefully introduced into a new syringe together with 2 ml HA (molecular weight 8 × 105, Artz®; Seikagaku Kogyo, Tokyo, Japan), and the cell suspension containing 7.0 × 106 labeled MSCs was injected into the medial compartment of the left knee joint of 7-month-old guinea pigs. For this purpose, an 18-gauge needle was inserted posterior to the medial edge of the patellar ligament, through the triangle formed by the epicondyle of the femur, the meniscal/tibial plateau, and the notch formed by their junction.

Subtyping of animal groups and histological samples The animals were divided into four treatment groups at random: PBS group (n = 15), HA-injected group (n = 15), PBS + MSC group (n = 15), and HA + MSC group (n = 15). The control groups were similar to the test groups with respect to age and weight. In each group, the animals were sacrificed at 1, 3, and 5 weeks post transplantation of cells and/or carrier. The proximal tibia was dissected out and prepared for examination.

Macroscopic examination The surface of the distal head of the femur and the tibial plateau were exposed. India ink (2 ml) was injected onto the joint surface using a syringe, and 1 minute later the surfaces were washed with physiological saline. The staining pattern of the cartilage surface was observed macroscopically. The gross findings were classified and scored into six grades according to the assessment suggested by Hayashi and colleagues [15]. The assessment was conducted by two independent examiners (HN, TM), who were blinded to each other's findings and to the treatment group assignment of the animals. Finally, the scores evaluated by these two examiners were averaged to obtain the overall score.

Histological and immunohistochemical examinations The proximal tibia was harvested and fixed in 10% buffered formaldehyde solution at 4°C for 48 hours. The samples were snap frozen in liquid nitrogen and then embedded in optimal cutting temperature compound. The frozen blocks were cut into sections 10 μm thick according to the methods described previously [16]. Serial sections 10 μm thick, 20 frontal sections in each knee, were prepared carefully in order to include the severely degenerated area not covered by meniscus. Sections from each animal were used for histological, immunohistochemical, and fluoroscopic analyses.

The sections were stained with H & E and safranin O using standard methods. We assessed the severity of knee OA using the modified Mankin criteria described by Armstrong and colleagues [17], expressed as the OA score. All sections were graded by three independent observers blinded to the treatment group, and the median score was used for statistical analysis.

The tissue sections were also stained with a primary polyclonal antibody, raised in rabbits against type II collagen (dilution 1:200; COSMO BIO, Tokyo, Japan), for 1 hour at room temperature. After rinsing in PBS, the tissue was incubated with biotinylated horse anti-rabbit IgG secondary antibody for 30 minutes at room temperature. Immunohistochemical staining was detected with VECTA STAIN ABC Reagent (Vectastain Elite Kit; Vector Laboratories, Burlingame, CA, USA), followed by staining with 3,3-diaminobenzidine tetrahydrochloride (Dojin Chemicals, Tokyo, Japan). In addition, to evaluate the location of collagen type II relative to the cells, other sections were incubated with the secondary antibody for goat anti-rat Alexa Flour® 568-conjugated antibody (1:250; Molecular Probes) for 1 hour at room temperature. These sections were counterstained with the nuclear marker 4',6-diamidino-2-phenylindole.

Cells labeled with CFDA-SE can be visualized by fluorescence microscopy using standard fluorescein filter sets. The approximate excitation and emission peaks of this product after hydrolysis are 492 nm and 517 nm. The labeled cells were quantified in 500 × 500 μm2 areas of tibial frontal section (original magnification, ×100).

Immunoblot analysis Immunoblot analysis was carried out with 15% SDS-PAGE. Total protein (80 μg/lane) extracted from the cartilage was transferred onto polyvinylidene difluoride membrane for 70 minutes using a semi-dry blot apparatus. The membrane was washed twice in PBS containing 0.05% Tween 20, blocked by 5% skimmed milk in PBS for 1 hour at room temperature, and then incubated with the collagen type I, collagen type II, or matrix metalloproteinase-13 primary antibody (dilution 1:20; COSMO BIO) overnight at 4°C, followed sequentially by anti-mouse IgG antibody and avidin-biotinylated peroxidase complex (for 30 minutes each). After triple washing in 0.1 M PBS, the membrane was incubated in enhanced chemiluminescence reagent for 1 minute, and then the antibody binding was visualized using a FluorChem™ 8000 System (Alpha Innotech Corporation, San Leandro, CA, USA). The size of each band was normalized to β-actin (lot number IMG-5142A, 1:1,000; Imgenex, San Diego, CA, USA).

Statistical analysis Data are expressed as the mean ± standard deviation. Differences between groups were examined for statistical significance using one-way analysis of variance followed by the Bonferoni/Dunn post-hoc paired test for comparison between groups. P < 0.05 denoted the presence of a significant difference between groups. The above tests were conducted using the Statistical Package for Social Sciences (version 11.0; SPSS Inc., Chicago, IL, USA).

Results

Assessment of xenogeneic transplantation of MSCs Clinically, all guinea pigs tolerated the cell injection, and there was no evidence of local inflammation, joint effusion, or unloading of the joint resulting from the cell treatment. Histologically, the frontal section of the entire knee joint showed no evidence of synovial hyperplasia (Figure 1A, B, C). Fluorescence examination showed labeled MSCs in the fibrillated cartilage at the medial tibial plateau (Figure 1D'). A few labeled cells were also detected in the synovial lining and medial meniscus (Figure 1B', C'), but no such cells were found in the lateral region of the articular cartilage and meniscus (Figure 1E').

Macroscopic findings No India ink staining was observed in the lateral tibial plateau of any specimen. In the PBS, HA, and PBS + MSC groups, the surfaces of the articular cartilage over the bare medial tibial plateau (not covered with the meniscus) were rough and strongly stained at 5 weeks post transplantation (Figure 2A, B, C). In the HA + MSC group, the surface of the medial plateau was relatively smooth and the staining intensity was weaker in the same time interval (Figure (Figure2D).2D). The macroscopic OA score was significantly lower in the HA + MSC group than in the other groups (Figure (Figure2E2E).

Histological findings The PBS and PBS + MSC groups showed depletion of chondrocytes (which extended to the transitional zone) and matrix fibrillation (which extended from the transitional zone to the radial zone) (Figure 3A, C). The safranin-O-stained matrix was reduced in all treatment groups with the exception of the HA + MSC group (Figure 3E, G). In the HA group, matrix fibrillation of the articular surface did not extend to the radial zone (Figure (Figure3B)3B) and weak matrix staining and cell depletion were observed (Figure (Figure3F).3F). The HA + MSC group showed large numbers of chondrocytes with cluster formations in the radial layer of the articular cartilage. Furthermore, the matrix around the cell clusters was strongly stained in safranin-O-stained sections at 5 weeks post transplantation (Figure 3D, H).

Immunohistochemical and immunoblot analyses The immunostaining for type II collagen was stronger and more extensive around the chondrocyte-like cells at 5 weeks in the HA + MSC group compared with the other three treatment groups (Figure (Figure4A).4A). Similarly, western blot analysis showed higher levels of type II collagen in the HA + MSC group, relative to the PBS group (Figure (Figure4B).4B). Weak bands of type I collagen were observed in every group, and there were no significant differences between the groups. The bands of matrix metalloproteinase-13 were weaker in the HA + MSC group, relative to the PBS and PBS + MSC groups (Figure (Figure4B).4B). Quantitative analysis confirmed these findings (Figure (Figure4C4C).

Fluorescent microscopic findings A few CFDA-SE-labeled cells were found within the cartilage at 1 week post transplantation of the PBS + MSC group (Figure (Figure5A).5A). The labeled cells gradually disappeared from the cartilage at 3 and 5 weeks post transplantation (Figure 5B, C). In contrast, the labeled cells appeared both adhered to the surface and scattered within the superficial and transitional layers of the cartilage at 1 week post transplantation in the HA + MSC group (Figure (Figure5D),5D), but they were more frequent in the transitional layer and showed a columnar arrangement at 3 and 5 weeks (Figure 5E, F). The number of labeled cells was higher in the HA + MSC group than in the PBS + MSC group at all the time periods (Figure (Figure5G).5G). These findings suggest that the CFDA-SE-labeled MSCs in HA migrated into the OA cartilage and were associated with the improved metachromasia and histology OA score.

Double immunofluorescence stained sections were evaluated for the distribution of residual chondrocytes, labeled MSCs, and type II collagen in the OA cartilage at 5 weeks post transplantation of HA + MSC injection. Immunostaining for type II collagen was observed in the matrix around residual chondrocytes in the radial layer at 5 weeks (Figure 6B, C), in addition to pericellularly and in the matrix between labeled MSCs in the transitional layer (Figure 6B, D). These findings indicated chondrogenic differentiation of the injected MSCs.

Discussion

MSCs can be isolated from a variety of mature tissues, and readily expand in culture without losing their multilineage differentiation potential [4,18,19]. Moreover, MSCs secrete a broad spectrum of bioactive molecules, collectively known as trophic factors [20]. Previous reports, however, have demonstrated that MSCs represent only 0.01 to 0.001% of the total nucleated cells within isolated bone marrow aspirates [4] and that the true identity of MSCs is often confused by various isolation and in vitro culture methods [21]. In this regard, preparation of large quantities of autologous MSCs for therapeutic use from small experimental animals is sometimes technologically difficult. Several groups reported recently that MSCs produce molecules that are directly involved in the regulation of the immune response and have immunosuppressive properties, which may be particularly important in allogeneic MSC transplantation [22,23]. In addition, human MSCs evade xenogeneic immune reactions and inhibit the inflammatory response in collagen-induced arthritis in mice [24]. For these reasons, we used commercially available human MSCs for xenogeneic MSC transplantation in this study. In the present study, the guinea pigs tolerated the cell injections well, and there was no evidence of local inflammation. Frontal histological section of the whole knee joint showed no evidence of synovial hyperplasia. Fluorescence microscopy also identified the presence of the labeled MSCs in the fibrillated cartilage at the medial tibial plateau. A few labeled cells were also detected in the synovial lining and medial meniscus.

In the present study, we evaluated the feasibility of intra-articular injection of MSCs suspended in HA for the treatment of knee OA. Our results at 5 weeks post transplantation showed histologically confirmed partial repair of the articular cartilage, compared with the three other treatment groups. Immunoblot analysis also showed increasing content of type II collagen and low levels of matrix metalloproteinase-13 in the repaired cartilage. In vivo tracing techniques using CFDA-SE labeling and fluorescence microscopy demonstrated that the intra-articularly injected MSCs in HA migrated throughout the osteoarthritic cartilage. Double immunofluorescence analysis also demonstrated strong, although partial, immunostaining of type II collagen around both residual chondrocytes and the injected MSCs after 5 weeks. These results imply intra-articular differentiation of the injected MSCs into chondrocytes and their subsequent proliferation. While HA is known to influence chondrocyte metabolism, the MSCs may produce trophic factors that could also stimulate the residual chondrocytes.

In our animal study, MSCs possibly produced trophic factors for residual chondrocytes, in addition to differentiating into chondrocytes in the presence of HA. While the biochemical conditions necessary for stimulation of chondrogenesis of MSCs in vitro have been defined [25], little is known about the differentiation of MSCs into chondrocytes and cartilage regeneration in vivo. Several studies investigated chondrogenesis of grafted MSCs within scaffolds in full-thickness cartilage defects in animal models [26,27]. The analyses in these studies showed improved tissue filling and increased matrix staining for type II collagen and proteoglycans 5 to 8 weeks after grafting. The studies' results showed that the transplanted cells secreted type II collagen and contributed to articular cartilage repair. Although simple injection of MSCs was not successful in the repair of cartilage in the present study, MSCs suspended in HA appeared to migrate and proliferate into the transitional layer - and differentiation of these cells into chondrocytes was observed at week 5 after injection. For this treatment group, there was partial repair of cartilage degeneration, both macroscopically and microscopically. The results thus suggest that HA is an important factor in MSC transplantation into the articular cartilage.

There are several advantages for the use of the MSC and HA mixture for intra-articular injection. HA exhibits biological affinity for MSC binding via the transmembrane receptor CD44, which facilitates cell migration through interaction with extracellular HA [28]. HA also increases the chondrogenic activity of MSCs [29]. Another study demonstrated that HA coats the surface of the articular cartilage and also locates among the collagen fibrils and sulfated proteoglycans within the cartilage [30]. Furthermore, in vitro experiments indicated that HA alone enhanced the rate of synovial cell migration, and that HA increased chondrocyte migration in the presence of basic fibroblast growth factor [31]. Considered together, our findings and the results of the above studies emphasize the importance of HA in facilitating the migration, adherence, and differentiation of MSCs onto the OA cartilage.

Previous studies used the MSC-HA combination for cartilage repair in surgically induced OA [9,10], but not for spontaneous OA as was carried out in the present study using the Hartley strain guinea pigs. We compared the difference between disease progression of Hartley guinea pigs knees, with and without transection of the anterior cruciate ligament [32]. Their results showed accelerated cartilage degeneration and higher levels of matrix metalloproteinase-13 and IL-1β, which is a key mediator for OA progression, in the anterior cruciate ligament transection model. These results indicated that the progression of OA may differ between surgically induced and spontaneous OA. In this regard, this animal model of spontaneous OA is considered more suitable for the evaluation of disease-modifying OA treatments.

Our study has certain limitations that should be considered when interpreting the results of the experiments. These include the use of nonphysiologic conditions for xenogeneic MSC transplantation, possible deterioration of the repaired cartilage after injection, a lack of ultrastructural analysis such as morphological evaluation by electron microscopy to clarify the in vivo chondrogenesis of MSCs in OA cartilage after the injection, a lack of re-examination of optimum timing of injection during OA progression, lack of a MSC population isolated from various types of adult mesenchymal tissues and various issues related to the transplantation of autologous or xenogeneic derived stem cells, and the use of HA only instead of including another compound with molecular weight similar to HA. Further studies are needed that include the above points. Should these issues be resolved satisfactorily, then intra-articular injection of MSCs suspended in HA could be a potentially useful therapeutic intervention for the treatment of spontaneous knee OA.

Conclusion

The results of the present study suggest that intra-articular injection of MSCs with HA is a potentially beneficial therapy for OA. HA exerts cell-binding and cytotactic effects, thus enhancing the migration and proliferation of MSCs. Although further examination of the long-term effects is necessary, this scaffold-free, minimally invasive treatment seems to retard the progression of spontaneous OA and stimulates the regeneration of articular cartilage.

Abbreviations

CFDA-SE: carboxyfluorescein diacetate succinimidyl ester; HA: hyaluronic acid; H & E: hematoxylin and eosin; IL: interleukin; MSC: mesenchymal stem cell; OA: osteoarthritis; PBS: phosphate-buffered saline.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

MS carried out most of the experiments, interpreted the data, and drafted the manuscript. KU contributed to study design and conception, analysis of data, and drafting of the manuscript. HN and TM contributed to analysis and interpretation of data and drafting of the manuscript. ARG and SW contributed to acquisition of data and drafting of the manuscript. SR contributed to acquisition of data and critical revision of the manuscript. HB contributed to study conception and critical revision of the manuscript. All authors read and approved the final manuscript.

Acknowledgements

The present work was supported in part by a grant-in-aid for general scientific research to TM from the Ministry of Education, Science and Culture of Japan (grant number 23791632).

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Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

Markus T. Berninger, Gabriele Wexel, Ernst J. Rummeny, Andreas B. Imhoff, Martina Anton, Tobias D. Henning,# and Stephan Vogt#

Abstract

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface 1. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential 2. In the last decades, several surgical treatment options have emerged and have already been clinically established 3-6.

Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface 3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.

New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation 9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone 11.

The sandwich-technique combines bone grafting with current approaches in Tissue Engineering 5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing 12.

Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity 11.

Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential 13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.

In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results 1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies 19-21 and even first human trials 22.

The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit's bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit's knee joint will be described.

Protocol

A. Preparation of a Donor Rabbit for the Isolation of Mesenchymal Stem Cells (Surgery Room)

• 1. Cells are isolated from male New Zealand White (NZW) rabbits at age of 4 months and approximately 3 kg body weight.
• 2. Induce anesthesia by propofol (10 mg/kg body weight i.v.) and sacrifice with sodium pentobarbital (100 mg/kg body weight i.v.).
• 3. Shave fur from hind limbs, back and belly with an electric clipper and vacuum the fur.
• 4. Disinfect the shaved area thoroughly with 70% ethanol.
• 5. Use blunt forceps, sharp scissors (or scalpel) and bone cutters for tissue and ligaments.
• 6. Make an incision along the cranial surface of the leg and calf.
• 7. Reflect skin and subcutaneous tissue by either sharp or blunt dissection.
• 8. Separate muscles and ligaments from tibia and femur. Keep cuts as close to the bone as possible to make a clean dissection. Do not separate femur from tibia at this time.
• 9. Cut through the hip joint to separate the head of the femur from the acetabulum.
• 10. Elevate the tibial-femoral complex.
• 11. Use the scalpel blade to scrape off any remaining soft tissue from the bones or rub the bones with sterile cloth tissues. At this point, femur and tibia are still connected.
• 12. Remove patella by cutting, then cut knee joint ligaments to separate bones finally.
• 13. Spray separated bones with 70% ethanol, let air dry and place each bone into a 50 ml centrifuge tube with cell culture medium (DMEM + 1% penicillin/streptomycin (Pen/Strep)) to keep them moist.
• 14. Now switch under a sterile cell culture laminar flow hood.

B. Flushing of Rabbit MSC from Bones and Expansion (Cell Culture Hood)

• 1. Collect bones from tubes and place them into 150 mm dishes using sterile forceps.
• 2. Remove both bone ends with a sterile saw and move pieces to new 150 mm dishes.
• 3. Fill a 10 ml syringe with medium (DMEM), attach an 18 gauge needle and insert into the opening of the bone marrow.
• 4. Then, rinse marrow cavity with medium to flush the bone marrow into the dish. Afterwards, rinse from the other end, if possible. If necessary, saw off more from the ends. If bone should break, just rinse the inside of the bone.
• 5. Aspirate cell medium suspension into the syringe and rinse the bone marrow repeatedly until suspension is free floating through the bone marrow cavity and no further bone marrow-clots appear.
• 6. Once bone marrow has been collected from all bones, disrupt the marrow clumps by passing through an 18 gauge needle: fill syringe with needle attached and force out into medium.
• 7. Afterwards, filter the suspension through a cell filter into a 50 ml tube. In order to prevent cell loss, wash culture dish 2x with 10 ml medium and filter as well.
• 8. Centrifuge suspension at 500 x g for 5 min at RT.
• 9. Remove supernatant and resuspend cell pellet in 10 ml medium (DMEM + 1% Pen/Strep).
• 10. Separate blood cells from Peripheral Blood Mononuclear Cells (PBMC) and mesenchymal stem cells (MSC) using a Biocoll Separating Solution.
• 11. Fill 5 ml of Biocoll Separating Solution into a 15 ml tube and carefully add 5 ml of cell-suspension on top and centrifuge at 800 x g for 20 min at RT (without brake).
• 12. Possible results see Figure 1: Being denser than Biocoll, red blood cells sediment to the bottom while PBMC and mesenchymal stem cells remain at the interface.
• 13. Carefully remove interface into a 15 ml tube and wash with 5 ml PBS.
• 14. Centrifuge as described in step 22, resuspend in 5 ml PBS and repeat 2-3x.
• 15. Then, centrifuge again at 350 x g for 10 min at RT (with brake).
• 16. Resuspend in 10 ml medium and count cells in a hemocytometer.
• 17. Plate cells at an initial seeding density of approximately 5 x 106 in 150 mm dishes.
• 18. After 2-3 days, remove non-adherent cells. You might have to rinse with PBS first in order to remove cell debris. Add fresh complete medium (DMEM + 10% Fetal Calf Serum (FCS) + 1% Pen/Strep) afterwards.
• 19. Feed cells every 3-4 days (Figure 2).
• 20. After 5-10 days, passage the cells for the first time.

C. Preparation of Fibrin Clots in vitro

• 1. At the day of implantation, release adherent cells from flasks/dishes by a 3 min-exposure to 0.25% trypsin-EDTA. Stop trypsinization by adding complete medium.
• 2. Distribute cells in a 50 ml falcon tube and wash them twice with PBS.
• 3. Determine cell viability and numbers by trypan blue staining.
• 4. Add 50,000 cells/microcentrifuge tube and collect pellets by centrifugation at 500 x g for 5 min at RT. Prepare a mastermix for at least one more clot as needed.
• 5. Resuspend the cell pellet in 17 μl PBS and mix 25 μl of the fibrinogen component of TISSUCOL-Kit with this 17 μl MSC suspension.
• 6. Take a sterile plate with pre-drilled holes (3x3.6 mm) in accordance to the drill holes in vivo(Figure 3).
• 7. First, inoculate 4 μl thrombin solution (500 IU/ml) into one hole, followed by immediate addition of 42 μl fibrinogen-cell-suspension and again 4 μl thrombin solution on top. Do not mix the suspension to avoid clotting in the pipette tip. First, the 50 μl volume of the pipetted fibrinogen-cell-suspension will protrude the rim of the pre-drilled holes without melting due to the surface tension. However, after complete clotting (after 60 min) the clot is contracting and fits into the pre-drilled hole.
• 8. Remove the clot carefully using a blunt forceps and place into a microcentrifuge tube with PBS. Prepare 2 clots/animal.
• 9. Take the clots to the surgery room.

D. Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

• 1. Induce anesthesia to rabbit (NZW, male, 3.5-4.0 kg body weight, 5-6 months old) by i.v. injection of propofol (10 mg/kg body weight).
• 2. Shave the knee to be operated on with an electric clipper and vacuum the fur. All the procedures named before are performed in a surgery preparation room to avoid contamination of the sterile environment of the operating room.
• 3. After intubation, maintain anesthesia with 1.5 mg/kg/min propofol and 0.05 mg/kg/min fentanyl intravenously. Monitor anesthesia by using capnography, pulse oximetry and pulse rate.
• 4. Disinfect the shaved knee thoroughly and cover the rest of rabbit with a sterile dressing.
• 5. Palpate the patella and perform a skin incision medial to the patella.
• 6. Open the knee joint by a medial parapatellar arthrotomy under sterile conditions. Try to avoid cutting any small superficial blood vessels.
• 7. Displace the patella laterally (Figure 4).
• 8. After inspection of the knee joint for any concomitant cartilage lesions or joint anomalies, create two osteochondral defects (3 mm deep, figure eight-shaped) in the trochlear groove with a sterile air operating
• power drill (3.6 mm in diameter) with a stop-device (Figure 5) .
• 9. Clean the defects and rinse them with sterile saline.
• 10. Prior to implantation, fill 20 μl of fibrin glue into the defects and distribute them evenly onto the bottom of the defect.
• 11. Then implant the clots press-fitted into the figure eight-shaped defect.
• 12. After clotting, relocate the patella within the trochlear groove and bend and stretch the knee a few times.
• 13. Displace the patella laterally once again and check if fibrinogen-cell-clots are still in place.
• 14. Replace the patella again and finish the operation with wound closure in layers with single button sutures (4-0 Vicryl) and a continuous cutaneous suture (4-0 Monocryl) (both with absorbable suture material).
• 15. Finally, seal the wound with a spray dressing permeable to water vapor.
• 16. For post-operative care, the wound is checked daily for 7 days. The rabbits receive for post-operative analgesia Carprofen 4 mg/kg s.c. every 24 hr (for 4 days) and Buprenorphin 0.03 mg/kg s.c. every 12 hr (for 2.5 days). A stabilization of the knee (e.g. dressing) is not necessary.

Representative Results

The described surgical technique permits a successful isolation and implantation of allogeneic mesenchymal stem cells into an artificial osteochondral defect. The experimental setup resulted in a successful integration of the implant into the surrounding cartilage.

The defect was filled by repair tissue with similar biomechanical properties and similar durability compared to the surrounding cartilage. The fibrin-cell-clot was prepared in vitro on a sterile plate with pre-drilled holes, which had the same size as the osteochondral defect (Figure 3). As a result, there were no clefts between the implanted fibrin clot and the surrounding cartilage, which would be a risk factor for premature degeneration or delamination (Figure 6). A basal healing of the repair tissue was ensured because the subchondral bone was penetrated, and thus worked against a shearing. Another important aspect was the stiffness of the repair tissue, which should match the healthy surrounding cartilage tissue to avoid an increased load on it and a possible premature degeneration. In our preliminary experiments (data not shown), we showed that after 12 weeks a sufficient rigidity could already be achieved. Moreover, an intact and homogeneous surface of the transplant was found, which reduced shear stress and possible implant damage (Figure 6).

Discussion

In recent years, the possibility of treating complex articular osteochondral defects - such as those resulting from osteochondritis dissecans, osteonecrosis and trauma - with Tissue Engineering approaches became more and more attractive. In the previously mentioned pathologic entities, tissue damage extends to the subchondral bone and involves two tissues characterized by different intrinsic healing capacities 1. There is an increasing interest in the role of subchondral bone for the pathogenic processes of osteochondral articular damage 11,23. The functional conditions of articular cartilage and its supporting bone are tightly connected. Injuries of either tissue adversely affect the mechanical environment as well as the homeostatic balance of the entire joint 24. Alterations in the osteochondral unit through mechanical disruption of joint motion, loose body formation, mechanical wear in the involved compartment and attrition of opposing surfaces may lead to an earlier onset and development of osteoarthritis 1,11. Therefore, tissue engineering approaches for the regeneration of osteochondral defects should be accompanied by an adequate restoration of the underlying subchondral bone in order to enhance the effective union with surrounding host tissues 2. Mesenchymal stem cells seem to be an ideal cell source to provide these specific requirements of osteochondral repair. The protocol highlights the promotion of bone and cartilage tissue restoration by potentially inducing the selective differentiation of the transplanted mesenchymal stem cells in osteogenic and chondrogenic lineages.

In comparison to chondrocytes, mesenchymal stem cells have several major advantages: they can be easily isolated from bone marrow, synovialis and fat tissue without any greater donor side morbidity. Mesenchymal stem cells do not differentiate during in vitro expansion and therefore can be culture-expanded in large numbers to treat large articular cartilage defects. Moreover, they seem to be immunosuppressive and - in response to appropriate stimuli - can differentiate into chondrocytes and osteocytes 25,26. Another remarkable advantage of mesenchymal stem cells displays their hypoimmunity, which means that allogeneic mesenchymal stem cells can be used without any sign of rejection reaction 27. Therefore, a cell-pool from one or two donor animals can be sufficient for all experiments. This reduces operation time and additional harm to the animals.

Several experiments showed promising results using fibrin glue for osteochondral repair 19,20. Usually, the inoculation of the fibrinogen-cell-suspension with thrombin solution has been described as a procedure done in situ, directly into the artificial osteochondral lesions. After a short period of time of pre-clotting (after a few minutes) the operation is usually finished by relocating the patella and wound closure.

In several pilot tests, we found out that for sufficient clotting of the fibrin-cell-suspension it takes more than 60 min. In situ - during the operation - it is hardly possible to wait more than 60 min for entire clotting. Moreover, by use of a sterile plate with pre-drilled holes simulating the osteochondral lesions, it was possible to show that the amount of fibrin glue, which was used to obviously fill the defect completely, was not enough due to shrinking of the hardening clot. This requires a higher volume of fibrin glue in advance in order to achieve a congruent and complete filling of the defect. Performing the preparation of the clot in vitro it is possible to easily adjust the construct shape to the appropriate size of the drilled defect and therefore, fill the osteochondral defect totally and congruently.

Additionally, an in vitro preparation of the cell clots prevents a leakage of the adhesive but (after only a few minutes) not fully hardened fibrin-cell-suspension. Therefore, it can be guaranteed that the initially intended volume will stay in the defect and will begin with cartilage integration and remodeling.

The described technique permits a standardized method for experimental stem cell research in the field of osteochondral repair. The protocol provides a reproducible way to isolate mesenchymal stem cells in order to re-implant them later in osteochondral cartilage defects of the rabbit's knee joint. Autologous chondrocytes have already been implanted into osteochondral defects in a fibrin-cell-model 19. Using an in vitro-model of clot preparation as well as mesenchymal stem cells instead of chondrocytes appears to be a more advantageous and promising new approach for remodeling and repair of osteochondral lesions.

Disclosures

The authors have nothing to disclose.

Acknowledgments

This project was funded by the German Research Association (grant HE 4578/3-1) and partially by the FP7 EU-Project “GAMBA” NMP3-SL-2010-245993.

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