Autologous chondrocyte implantation (ACI) is a cell-based therapy that has been used clinically for over 20 years to treat cartilage injuries more efficiently in order to negate or delay the need for joint replacement surgery. In this time, very little has changed in the ACI procedure, but now many centres are considering or using alternative cell sources for cartilage repair, in particular mesenchymal stem cells (MSCs). In this study, we have tested the chondrogenic potential of donor-matched MSCs derived from bone marrow (BM), infrapatellar fat pad (FP), and subcutaneous fat (SCF), compared to chondrocytes. We have confirmed that there is a chondrogenic potency hierarchy ranging across these cell types, with the most potent being chondrocytes, followed by FP-MSCs, BM-MSCs, and lastly SCF-MSCs. We have also examined gene expression and surface marker profiles in a predictive model to identify cells with enhanced chondrogenic potential. In doing so, we have shown that Sox-9, Alk-1, and Coll X expressions, as well as immunopositivity for CD49c and CD39, have predictive value for all of the cell types tested in indicating chondrogenic potency. The findings from this study have significant clinical implications for the refinement and development of novel cell-based cartilage repair strategies.
Autologous chondrocyte implantation (ACI) for the treatment of focal chondral and/or osteochondral lesions has changed very little since its inception [
Mesenchymal stem cells (MSCs) isolated from the bone marrow (BM-MSCs) have been used in several clinical trials as an alternative cell source for use in cell therapies to treat cartilage injuries and osteoarthritis [
An important factor to consider when comparing and contrasting the properties of different cell types is the “donor impact” as donor demographics, such as age and gender, are factors which are known to affect cell proliferation and differentiation capacity [
In this study, we have examined 4 different cell types (chondrocytes, BM-MSCs, FP-MSCs, and SCF-MSCs) and tested the chondrogenic potential of each population of cells. This study compares donor-matched cell types and was designed to establish the impact of tissue source and donor on chondrogenic differentiation capacity and to continue the process of establishing a marker panel indicative of chondrogenic potency and likely clinical success. Such marker(s) could be screened for and used in the selection of a particular cell type and/or subpopulation of cells with enhanced chondrogenic capability prior to treatment. We envisage that taken together this information could significantly improve the success of cell-based therapies for cartilage injuries and perhaps even lead to the development of novel individualised treatments for cartilage repair.
All samples were obtained after patients had provided written informed consent; favourable ethical approval was given by the National Research Ethics Service (11/NW/0875) and all experiments were performed in accordance with relevant guidelines and regulations. Donor-matched samples of cartilage, BM, FP, and SCF were obtained from 5 patients (2 males and 3 females, ages 67–81 years) undergoing total knee replacement (TKR) surgery (Table
Donor demographics.
ID | Gender | Age (years) | Pathology |
---|---|---|---|
Donor 1 | Male | 71 | OA with extensive joint degeneration |
Donor 2 | Female | 67 | OA with loss of joint space |
Donor 3 | Female | 75 | Patellofemoral OA and loss of joint space in medial compartment |
Donor 4 | Female | 81 | OA |
Donor 5 | Male | 74 | OA with joint stiffness |
Macroscopically normal articular cartilage was excised from the femoral condyles of patients undergoing TKR. Cartilage tissue was weighed, minced into small pieces with a sterile scalpel, and digested in collagenase type II (250 IU/mg dry weight, Worthington, New Jersey, USA) for 16 hours at 37°C. The resulting suspension was passed through a 40
Bone marrow aspirates and bone chips were obtained from the tibial plateau of patients undergoing TKR. Bone marrow was first diluted with an equal volume of phosphate buffered saline (PBS, Life Technologies) then split between two 50 mL tubes, layered onto 10 mL of Lymphoprep
Human FP and SCF tissue samples were obtained from patients and processed within 2 hours of receipt from the operating theatre. The FP was dissected from the innermost zone, to avoid contamination with synovium derived cells as described previously [
After trypsinisation at passages 3-4,
Flow cytometry was used to assess the immunoprofile of chondrocytes and MSCs prior to chondrogenic differentiation. Cells at passages 3-4 were harvested, pelleted, and resuspended in 2% bovine serum albumin (BSA, Sigma-Aldrich) in PBS. FC receptors were blocked for 1 h at 4°C using 10% (v/v) human IgG (Grifols, Barcelona, Spain) in 2% (v/v) BSA in PBS (immunobuffer). The cells were then washed with immunobuffer and centrifuged (350
The chondrogenic potential of all cultured cell populations was assessed at passage 4 using an established 3D pellet culture system [
Pellets were digested using papain to release GAGs and DNA. A digestion buffer consisting of 50 mM sodium phosphate (BDH), 2 mM EDTA (Sigma-Aldrich), and 20 mM N-acetyl cysteine (BDH) was prepared and the pH adjusted to 6. Papain (Sigma-Aldrich) was added to the digestion buffer to reach a final concentration of 125
The dimethylmethylene blue (DMMB) assay was used to quantitate GAGs [
The PicoGreen® fluorescence assay (Invitrogen) was used to quantitate the amount of double stranded DNA in solution and was conducted according to the manufacturer’s instructions. Fluorescence was read on a plate reader configured to excitation = 480 nm and emission = 520 nm. The normalisation of GAG content in chondrogenic pellets was achieved by dividing the total GAG content of a given pellet by the DNA content of that same pellet.
Pellets were cryosectioned (7
The Shapiro-Wilk normality test was used to assess the distribution of quantitative data. A one-way ANOVA with Bonferroni’s multiple comparisons test was used to test for significant differences between cell types with regard to gene expression, immunoprofile, GAG quantitation, and histological scores for chondrogenic pellets. Pearson’s correlation coefficients were determined for gene-gene expression analyses and chondrogenic assessments (GAG quantitation and histological analyses). Multilevel modelling was conducted to determine whether gene expression and cell surface marker positivity were predictors of chondrogenic outcome as measured by GAG content of the pellet and histological scoring. In these models, cell source, gene expression, and cell surface marker positivity were considered as fixed effects, while the donor was considered as a random effect. The donor effect was determined using Wald’s tests. Graphs are shown as means ± standard deviation, with statistical significance considered at
Perhaps not too surprisingly, of the cell types tested in this donor-matched study, the chondrogenic potency genes (Sox-9, Coll II, aggrecan, and FRZB) were consistently expressed at the highest levels in culture expanded chondrocytes. Further, chondrocytes demonstrated the lowest expression profiles for the hypertrophic genes tested (Alk-1 and Coll X). Of the MSC populations that we have examined, BM-MSCs displayed chondrogenic and hypertrophic profiles that most closely resembled those of culture expanded chondrocytes. In contrast, the adipose sources of MSCs investigated (FP-MSCs and SCF-MSCs) were least like culture expanded chondrocytes and demonstrated the lowest chondrogenic potency and the highest hypertrophic gene expression profiles. SCF-MSCs expressed Alk-1 at significantly higher levels than chondrocytes and BM-MSCs (
The expression of chondrogenic and hypertrophic genes in monolayer cell populations prior to chondrogenesis. ((a)–(f)) Chondrocytes (Ch), bone marrow MSC (BM), fat pad MSC (FP), and subcutaneous fat MSC (SCF). Data shown are the means ± the standard deviation of triplicate runs and 5 donors for each cell population. One-way ANOVA and
Gene expression associations for all of the cell types examined in this study were tested using Pearson’s correlation coefficient analyses and are presented in a correlation matrix (Figure
Flow cytometry analyses revealed immunopositivity for the MSC markers CD73, CD90, and CD105 for all of the populations of cells examined, but to varying levels. FP and SCF derived MSCs adhered to ISCT criteria (i.e., >95% positive); chondrocytes and BM-MSCs also adhered to ISCT criteria for CD90 and CD105 positivity but were <95% positive for CD73. All of the cell populations tested were <2% positive for CD19, CD45, and HLA-DR, in line with ISCT criteria. Some positivity was recorded in all of the cell populations tested for CD34 with high levels (62.2–74.4% positivity) seen in the adipose derived MSCs, which also adheres to ISCT [
Immunoprofiles for MSC markers and putative chondrogenic potency markers of culture expanded cells prior to chondrogenesis. (a) ISCT MSC immunoprofiles. Immunoprofiles for the putative chondrogenic markers CD49c (b), CD166 (c), and CD39 (d). Flow cytometry was used to detect the percentage of positive cells for each marker on monolayer cell populations of chondrocytes (Ch), bone marrow MSC (BM), fat pad MSC (FP), and subcutaneous fat MSC (SCF) prior to chondrogenesis. Data shown are the means
After 28 days of differentiation, donors demonstrated variability in chondrogenic capacity across the cell types tested. When results from individual donors were examined, chondrocytes consistently produced chondrogenic pellets in terms of GAG quantitation and histological analyses, but the propensity for MSCs to undergo chondrogenic differentiation was variable between individuals. GAG/DNA analyses appeared to match the histological findings noted for each patient: that is, larger pellets with prominent matrix metachromasia had the highest levels of GAGs measured (Figure
Chondrogenic assessments of pellet cultures between donors. (a) Production of GAG/DNA in pellet cultures from chondrocytes (Ch), bone marrow MSC (BM), fat pad MSC (FP), and subcutaneous fat MSC (SCF). GAGs were measured after chondrogenic differentiation using the DMMB assay and normalised to the DNA content of pellets; each donor is represented in individual graphs. Data shown are the means
Chondrogenic assessments of pellet cultures across cell types. (a) Production of GAG/DNA in pellet cultures from chondrocytes (Ch), bone marrow MSC (BM), fat pad MSC (FP), and subcutaneous fat MSC (SCF) for all donors combined. (b) Chondrogenic histology scores for Ch, BM, FP, and SCF for all donors combined. (c) Mean chondrogenic pellet diameter (
Multilevel modelling analysis was performed in an effort to identify chondrogenic potency predictors prior to chondrogenic differentiation. This analysis demonstrated that Alk-1 expression and CD166 immunopositivity both negatively associated with GAG quantitation in pellet cultures. However, CD49c and CD39 expression positively associated with GAG quantitation and histological score, respectively. Sox-9 expression positively associated with chondrogenic histological score, whereas expressions of the hypertrophic genes Coll X and Alk-1 were shown to negatively associate (Table
Multilevel modelling.
GAG/DNA | Chondrogenic histology score | |||||
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Coefficient | 95% CI |
|
Coefficient | SE (95% CI) |
|
|
Sox-9 | −5.6 | −11.7, 0.5 | 0.07 |
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|
|
Coll II | 22.3 | −7.8, 52.4 | 0.14 | 3.96 | −0.16, 8.08 | 0.06 |
Aggrecan | −0.003 | −0.2, 0.2 | 0.97 | −0.019 | −0.04, 0.004 | 0.10 |
FRZB | 33.1 | −27.4, 93.6 | 0.3 | 0.86 | −7.46, 9.18 | 0.83 |
Coll X | −0.01 | −0.02, 0.003 | 0.12 |
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|
Alk-1 |
|
|
|
|
|
|
CD49c |
|
|
|
−0.04 | −0.02, −0.01 | 0.63 |
CD166 |
|
|
|
0.01 | −0.04, 0.034 | 0.16 |
CD39 | −0.02 | 0.01, 0.4 | 0.78 |
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Significant associations are indicated in bold.
Further multilevel model analysis showed that cell type (but not donor source) had a significant impact on the predictive aspect of gene expression for both of the chondrogenic assessments tested, that is, GAG quantitation (
In recent years MSCs isolated from BM, FP, and SCF have been compared and contrasted extensively in
Following an established
The multilevel modelling analyses performed in this study have allowed us to explore the relationships between putative chondrogenic potency markers (gene expression and surface marker profiles) and chondrogenic outcome based on combined data from each cell source tested, while simultaneously examining the potential influence of donor and cell type. However, we have not yet verified whether the predictive factors for chondrogenesis that we have identified for combined data are present if only individual cell types are analysed as we believe that the small donor size precludes this type of analysis in the present study. We should of course be cautious when interpreting any analyses derived from a small donor sample size and we acknowledge this as a limitation of the study. Nonetheless, our multilevel modelling has revealed that the expressions of Alk-1 and Coll X are negatively associated with chondrogenic potential in terms of histology scores and for Alk-1 expression, as well as the GAG content of chondrogenically induced pellet cultures. In contrast, Sox-9 expression prior to chondrogenesis positively correlated with histological pellet scores. Some of these gene associations match a previous report comparing FP-MSCs and SCF-MSCs [
In addition, our multilevel analysis indicates that CD49c and CD39 immunopositivity positively predicts GAG production and histological score, respectively, in cell pellets, with no significant difference observed between donors. Other studies have shown that CD49c positivity on chondrocytes and CD39 positivity on synovium derived MSCs are associated with increased
We have demonstrated the chondrogenic predictive value of high levels of Sox-9 and low levels of collagen type X or Alk-1 expression as well as immunopositivity for CD49c and CD39 in a combined data analysis of chondrocytes, BM-MSCs, FP-MSCs, and SCF-MSCs. Further individual analyses on larger donor cohorts will be required to validate these findings for individual cell types before these predictive factors could be used as selection criteria prior to the transplantation or banking of each cell type in the treatment of cartilage injuries. We have also shown, using donor-matched samples, that cell type significantly influences the chondrogenic potency of the MSC sources examined in this study; we have demonstrated that MSCs sourced from the infrapatellar fat pad of the knee or bone marrow provide the “next best” alternative to chondrocytes, in terms of
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors are grateful to the Engineering and Physical Sciences Research Council Centre for Doctoral Training and Arthritis Research UK (Grants 20253, 19429, and 18480) for supporting this work and to Miss Jade Perry for contributing to some of the chondrogenic data analysis described.