Review of Caplan (1991) on cell-based therapeutic technology using Mesenchymal Stem Cells.

This classic discusses the original 1991 publication 'Mesenchymal Stem Cells (MSCs)' by Dr. Caplan on the emergence of a new therapeutic technology of self-cell repair using MSCs. After the original classic publication, a large number of methods to regenerate injured tissue have been reported. Currently, MSCs are used clinically to repair articular cartilage defects, liver cirrhosis, cerebral infarction, spinal cord injury, graft-versus-host disease and others. As a result, MSCs are considered one of the most important cell sources for regenerative medicine. An MSC has been demonstrated to be a multipotent stem cell in cell culture and was thought to contribute to the regeneration of injured tissue at transplant sites, but recently, the concept of MSCs has changed such that they are now referred to as "medicinal signaling cells," owing to their often indirect effects on tissue repair and regeneration. Regardless of the name, either mesenchymal stem cells or medicinal signaling cells, MSCs will be used to regenerate injured tissue more widely in the near future.

Induction of chondrogenesis with a RANKL-binding peptide, WP9QY, in vitro and in vivo in a rabbit model.

WP9QY (W9) is a receptor activator of nuclear factor-κB ligand (RANKL)-binding peptide that inhibits osteoclastogenesis by blunting the RANKL-RANK interaction, and also increases osteoblastogenesis via RANKL reverse signaling. W9 has dual effects on osteoclasts and osteoblasts; however, it is unknown whether the peptide has an effect on chondrocytes. Here, we report that W9 induces proliferation and differentiation of chondrocytes in vitro and repairs full-thickness articular cartilage defects in vivo. W9 stimulated chondrocyte differentiation in a two-dimensional (2D) culture of human mesenchymal stem cells (hMSCs), and transforming growth factor ß3 (TGF-ß3) showed synergistic effects with W9 on chondrogenesis. W9 enlarged the size of 3D pellet cultures of hMSCs and produced chondrocyte-specific matrices, especially in combined treatment with TGF-ß3. The peptide also stimulated proliferation of hMSCs with induction of expression of chondrogenesis-related genes. Several RANKL inhibitors had no effect on chondrocytic differentiation. RANKL-knockdown experiments showed that W9 did not induce chondrogenesis through RANKL, but did induce osteoblastogenesis through RANKL. Intraarticular injection of W9 resulted in significant repair of full-thickness articular cartilage defects in rabbits. Taken together, these results suggest that W9 ameliorates the articular cartilage defects by increasing the volume of cartilaginous matrices with accompanying induction of proliferation and differentiation of chondrocytes via mechanisms independent of RANKL inhibition and RANKL reverse signaling. Since no pharmaceuticals are clinically available for treatment of cartilage damage such as osteoarthritis, our findings demonstrate the potential of W9 to address the unmet medical needs.

Transplantation of autologous bone marrow-derived mesenchymal stem cells under arthroscopic surgery with microfracture versus microfracture alone for articular cartilage lesions in the knee: A multicenter prospective randomized control clinical trial.

INTRODUCTION: To investigate the efficacy of the transplantation of autologous bone marrow-derived mesenchymal stem cells (BMSCs) under arthroscopy with microfracture (MFX) compared with microfracture alone. METHODS: Eleven patients with a symptomatic articular cartilage defect of the knee were included in the study. They were randomized to receive BMSCs with MFX (cell-T group, n=7) or MFX alone (control group, n=4). Clinical results were evaluated using International Knee Documentation committee (IKDC) knee evaluation questionnaires and the Knee Injury and Osteoarthritis Outcome Score (KOOS) before and 48 weeks after surgery. Quantitative and qualitative assessments of repair tissue were carried out at 48 weeks by T2 mapping of magnetic resonance images (MRIs) and the magnetic resonance observation of cartilage repair tissue (MOCART) scoring system with follow-up MRI. RESULTS: No significant differences between preoperative and postoperative IKDC and KOOS were observed in the cell-T or control group. However, forty-eight weeks after surgery, the cell-T group showed a trend for a greater KOOS QOL score compared with the control group (79.4 vs. 39.1, respectively; P=0.07). The T2 value did not differ significantly between the two groups, but the mean MOCART score was significantly higher in the cell-T group than in the control group (P=0.02). CONCLUSIONS: Compared with MFX alone, BMSC transplantation with MFX resulted in better postoperative healing of the cartilage and subchondral bone as determined by the MOCART score. Clinically, BMSC transplantation with MFX gave a higher KOOS QOL score after 48 weeks.

Local administration of WP9QY (W9) peptide promotes bone formation in a rat femur delayed-union model.

The WP9QY peptide (W9) consists of nine amino acids. It binds to RANKL and blocks RANKL-induced increases in bone resorption and osteoclastogenesis. W9 has a unique effect on the coupling mechanism between osteoclasts and osteoblasts, which promotes bone formation while working to suppress bone resorption. In this study, with the aim of clinical application of W9 for fracture treatment, we aimed to clarify the bone repair-promoting effect of W9 when administered locally to a rat femur model of delayed union. Using Sprague-Dawley rats, a model of delayed union was created in the right femur by cauterizing the periosteum. Injection of W9 (1 mg in 100 µl) or phosphate-buffered saline (PBS) (100 µl) at the fracture site was performed at the operation and every week thereafter until death (sacrifice). The bone union rate was 14% in the PBS group and 57% in the W9 group at 8 weeks postoperatively. The X-ray score of the W9 group was significantly higher than that of the PBS group at 8 weeks postoperatively. When bone morphometry was analyzed by micro-computed tomography (CT), total callus volume (TV) and mineralized callus bone volume (BV) were measured. TV showed no significant difference between the two groups, but BV/TV was significantly higher in the W9 group. This finding suggests that local administration of W9 can promote bone maturation from callus and can be considered to contribute to fracture healing. These results reveal that W9 has an effect on fractures of promoting healing and could be applied as a fracture treatment.

Quality Evaluation of Human Bone Marrow Mesenchymal Stem Cells for Cartilage Repair.

Quality evaluation of mesenchymal stem cells (MSCs) based on efficacy would be helpful for their clinical application. In this study, we aimed to find the factors of human bone marrow MSCs relating to cartilage repair. The expression profiles of humoral factors, messenger RNAs (mRNAs), and microRNAs (miRNAs) were analyzed in human bone marrow MSCs from five different donors. We investigated the correlations of these expression profiles with the capacity of the MSCs for proliferation, chondrogenic differentiation, and cartilage repair in vivo. The mRNA expression of MYBL1 was positively correlated with proliferation and cartilage differentiation. By contrast, the mRNA expression of RCAN2 and the protein expression of TIMP-1 and VEGF were negatively correlated with proliferation and cartilage differentiation. However, MSCs from all five donors had the capacity to promote cartilage repair in vivo regardless of their capacity for proliferation and cartilage differentiation. The mRNA expression of HLA-DRB1 was positively correlated with cartilage repair in vivo. Meanwhile, the mRNA expression of TMEM155 and expression of miR-486-3p, miR-148b, miR-93, and miR-320B were negatively correlated with cartilage repair. The expression analysis of these factors might help to predict the ability of bone marrow MSCs to promote cartilage repair.

High titer of anti-citrullinated peptide antibody is a risk factor for severe carotid atherosclerotic plaque in patients with rheumatoid arthritis: the TOMORROW study.

AIM: Cardiovascular disease is one of the complications of rheumatoid arthritis (RA). We researched the morbidity and severity of existing carotid atherosclerosis plaque and associated risk factors in patients with RA. METHOD: This study included 413 participants, including 208 patients with RA and 205 age- and sex-matched healthy volunteers. Carotid ultrasound, clinical data collection and assessment of cardiovascular risk factors were performed. Atherosclerotic plaque was defined as an intima-media thickness ≥ 1.1 mm. Severity of plaque was assessed by plaque score, defined as the sum of the maximal thickness of all plaques in bilateral carotid arteries. RESULTS: Data were analyzed from 200 patients with RA and 202 controls. Carotid plaque was observed more frequently in patients with RA than controls (47.0 vs. 36.1%, P = 0.027). Moreover, plaque score was significantly higher in RA patients (P = 0.032). In logistic regression analysis, RA represented an independent risk factor for the presence of plaque (adjusted odds ratio, 1.68; 95% confidence interval, 1.03-2.74). Comparing RA patients with and without plaque, anti-cyclic citrullinated peptide (anti-CCP) antibodies titer was significantly higher in patients with plaque (315.8 ± 454.1 U/mL) than in patients without (165.7 ± 281.1 U/mL; P = 0.005). Moreover, multiple linear regression analysis clarified that anti-CCP antibody titer was associated with plaque score in patients with RA. CONCLUSION: High prevalence of any carotid plaques and severe carotid plaques were more frequent in patients with RA. High titer of anti-CCP antibodies represented a risk factor for severe carotid atherosclerotic plaque in patients with RA.

Effect of epigallocatechin-3-gallate on the increase in type II collagen accumulation in cartilage-like MSC sheets.

With the aim to increase type II collagen content in the scaffold-free cartilage-like cell sheet using human bone marrow mesenchymal stem cells, we examined the effect of epigallocatechin-3-gallate (EGCG) addition to the chondrogenic medium for the cell sheet culture. The addition of EGCG (10 µM) increased the content of type II collagen 2-fold, while the addition did not markedly change the expression level of the genes encoding type II collagen and Sox 9. The reactive oxygen species level in the cells in cell sheets was thought to be too low to suppress the accumulation of type II collagen. On the other hand, the addition of EGCG markedly decreased both the matrix metalloproteinase-13 concentration in the supernatant of cell sheet culture and the type II collagen degradation activity in that supernatant. Taken together, EGCG may enhance the accumulation of type II collagen by suppressing type II collagen degradation.

Loss of lean body mass affects low bone mineral density in patients with rheumatoid arthritis - results from the TOMORROW study.

OBJECTIVES: Osteoporosis is one of the complications for patients with rheumatoid arthritis (RA). Rheumatoid cachexia, the loss of lean body mass, is another. However, the relationship between decreased lean body mass and reduced bone mineral density (BMD) in patients with RA has not been well studied. METHODS: This study included 413 participants, comprising 208 patients with RA and 205 age- and sex-matched healthy volunteers. Clinical data, BMD, bone metabolic markers (BMM) and body composition, such as lean body mass and percent fat, were collected. Risk factors for osteoporosis in patients with RA including the relationship BMD and body composition were analyzed. RESULTS: Patients with RA showed low BMD and high BMM compared with controls. Moreover, lean body mass was lower and percent fat was higher in patients with RA. Lean body mass correlated positively and percent fat negatively with BMD. Lean body mass was a positive and disease duration was a negative independent factor for BMD in multivariate statistical analysis. CONCLUSION: BMD and lean body mass were significantly lower in patients with RA compared to healthy controls. Lean body mass correlated positively with BMD and decreased lean body mass and disease duration affected low BMD in patients with RA. TRIAL REGISTRATION: [UMIN Clinical Trials Registry, http://www.umin.ac.jp/ctr/ , UMIN000003876].

Irx3 and Bmp2 regulate mouse mesenchymal cell chondrogenic differentiation in both a Sox9-dependent and -independent manner.

Sox9, a master regulator of cartilage development, controls the cell fate decision to differentiate from mesenchymal to chondrogenic cells. In addition, Sox9 regulates the proliferation and differentiation of chondrocytes, as well as the production of cartilage-specific proteoglycans. The existence of Sox9-independent mechanisms in cartilage development remains to be determined. Here, we attempted to identify genes involved in such putative mechanisms via microarray analysis using a mouse chondrogenic cell line, N1511. We first focused on transcription factors that exhibited upregulated expression following Bmp2 treatment, which was not altered by subsequent treatment with Sox9 siRNA. Among these, we selected positive regulators for chondrogenesis and identified Iroquois-related homeobox 3 (Irx3) as one of the candidate genes. Irx3 expression gradually increased with chondrocyte terminal differentiation in a reciprocal manner to Sox9 expression, and promoted the chondrogenic differentiation of mesenchymal cells upon Bmp2 treatment. Furthermore, Irx3 partially rescued impaired chondrogenesis by upregulating the expression of epiphycan and lumican under reduced Sox9 expression. Finally, Irx3 was shown to act in concert with Bmp2 signaling to activate the p38 MAPK pathway, which in turn stimulated Sox9 expression, as well as the expression of epiphycan and lumican in a Sox9-independent manner. These results indicate that Irx3 represents a novel chondrogenic factor of mesenchymal cells, acts synergistically with Bmp2-mediated signaling, and regulates chondrogenesis independent of the transcriptional machinery associated with Sox9-mediated regulation.

Effects of agitation rate on aggregation during beads-to-beads subcultivation of microcarrier culture of human mesenchymal stem cells.

With the aim to utilize human mesenchymal stem cells (hMSCs) grown in large scale for regenerative medicine, effects of agitation rate on aggregation during beads-to-beads subcultivation of microcarrier culture of hMSCs were studied. hMSCs could attach and grew on surface-type microcarriers of Cytodex 1, whereas almost no cell elongation and growth were observed on porous type microcarriers of Cytopores. The percentages of aggregated Cytodex 1 microcarriers at an agitation rate of 60 and 90 rpm were lower than that at 30 rpm, which was the lowest agitation rate necessary for the suspension of Cytodex 1 microcarriers, and the cells grew fastest at 60 rpm. hMSC could be subcultivated on Cytodex 1 by the beads-to-beads method at both 30 and 60 rpm without trypsinization. However, agitation at 60 rpm resulted in a markedly lower percentage of aggregated microcarriers not only before but also after subcultivation. The percentages of CD90- and CD166-positive cells among cells grown on Cytodex 1 at 60 rpm (91.5 and 87.6 %) were comparable to those of cells grown in the pre-culture on dishes. In conclusion, hMSCs could be subcultivated on Cytodex 1 by beads-to-beads method maintaining the expressions of the cell surface antigens CD90 and CD166, while adjusting agitation rate could decrease the microcarrier aggregation.

Circulating nucleated peripheral blood cells contribute to early-phase meniscal healing.

The purpose of this study was to assess how peripheral blood cells (PBCs) contribute to meniscus repair, using a parabiotic rat model. Wild-type (WT) and green fluorescent protein (GFP) transgenic rats were conjoined at the torso. After 4 weeks, the anterior part of the medial meniscus of both groups of rats was removed. At 1, 2, 4, 8 and 12 weeks post-meniscectomy, repaired tissue was evaluated using stereomicroscopy, histology with toluidine blue staining, and immunofluorescence microscopy. Stereomicroscopic observations and confocal laser microscopy revealed that a high number of GFP-positive cells were present in the repaired meniscus of WT rats 1 week post-meniscectomy, and the number of GFP-positive cells decreased over time. Based on blood chimerism, the ratios of PBCs in the repaired meniscus were 20.5 ± 2.3% at 1 week, 8.3 ± 0.9% at 2 weeks, 4.4 ± 0.9% at 4 weeks, 2.1 ± 0.9% at 8 weeks, and 0.5 ± 0.4% at 12 weeks, post-meniscectomy. Histologically, fibrochondrocytes were observed in the repaired meniscus of WT rats after 4 weeks, some of which were GFP-positive. The chondrogenic marker, type II collagen, was merged within the PBCs in the repaired tissue. However, type-II-collagen-positive cell ratio and metachromasia in the repaired meniscus were not equivalent in normal meniscal tissue. This indicated that PBCs were present within the repaired meniscus at an early phase, replacing the excised meniscal cells, suggesting PBCs contributed to meniscal healing. The tissue repair contribution by these cells decreased at later phases. Copyright © 2014 John Wiley & Sons, Ltd.

Magnetic Targeted Delivery of Induced Pluripotent Stem Cells Promotes Articular Cartilage Repair.

Cartilage regeneration treatments using stem cells are associated with problems due to the cell source and the difficulty of delivering the cells to the cartilage defect. We consider labeled induced pluripotent stem (iPS) cells to be an ideal source of cells for tissue regeneration, and if iPS cells could be delivered only into cartilage defects, it would be possible to repair articular cartilage. Consequently, we investigated the effect of magnetically labeled iPS (m-iPS) cells delivered into an osteochondral defect by magnetic field on the repair of articular cartilage. iPS cells were labeled magnetically and assessed for maintenance of pluripotency by their ability to form embryoid bodies in vitro and to form teratomas when injected subcutaneously into nude rats. These cells were delivered specifically into cartilage defects in nude rats using a magnetic field. The samples were graded according to the histologic grading score for cartilage regeneration. m-iPS cells differentiated into three embryonic germ layers and formed teratomas in the subcutaneous tissue. The histologic grading score was significantly better in the treatment group compared to the control group. m-iPS cells maintained pluripotency, and the magnetic delivery system proved useful and safe for cartilage repair using iPS cells.

Synergistic effect of ascorbic acid and collagen addition on the increase in type 2 collagen accumulation in cartilage-like MSC sheet.

Aiming to increase the content of type 2 collagen in scaffold-free cartilage-like cell sheets prepared using human bone marrow mesenchymal stem cells, the effect of several kinds of additives in a chondrogenic medium was investigated. Addition of ascorbic acid 2 phosphate (VCP) at a high concentration (250 µg/ml) and type 1 atelocollagen (5 µg/ml) increased the accumulation of type 2 collagen by fourfold and twofold, respectively. On the other hand, an antioxidant, glutathione showed no such effect. The synergistic effect of VCP and type 1 atelocollagen resulted in an eightfold increase in the accumulation level of type 2 collagen. Furthermore, the gene expression level of type 2 collagen increased and that of matrix metalloproteinase-13 (MMP-13) decreased to approximately one-third of the control. The increase in type 2 collagen accumulation in the scaffold-free cartilage-like cell sheet might be due to not only the enhancement of the synthesis but also the suppression of the degradation of type 2 collagen by MMP-13.

Effect of epigallocatechin-3-o-gallate and quercetin on the cryopreservation of cartilage tissue.

The effects of epigallocatechin-3-o-gallate (EGCG) and quercetin on the contents of extracellular matrix (ECM) in porcine cartilage at 4 °C were investigated. The addition of quercetin at 0.01 mM for the incubation of porcine cartilage disks at 4 °C for 2 week could suppress the decrease in ECM and the compliance of the disks, markedly greater than those of EGCG (1.0 mM).

High frequency of vertebral fracture and low bone quality in patients with rheumatoid arthritis-Results from TOMORROW study.

OBJECTIVES: Osteoporosis is one of the complications in patients with rheumatoid arthritis (RA). In this study, we researched the morbidity of existing vertebral fractures and the risk factors for vertebral fractures in patients with RA. METHODS: This study included 413 participants, 208 patients with RA, and 205 age- and sex-matched controls without RA. Clinical data, radiographic assessment of vertebral fracture from T4 to L4 in thoracic and lumber spine, bone mineral density (BMD), and bone metabolic markers (BMM) were analyzed. RESULTS: Vertebral fractures were observed more frequently, severe and multiple in patients with RA. In the logistic regression analysis, age (adjusted odds ratios (OR): 1.07, 95% confidence interval (CI): 1.04-1.09) and RA (adjusted OR: 1.72, 95% CI: 1.04-2.83) were risk factors for existing vertebral fracture. Moreover, two bone matrix-related markers, undercarboxylated osteocalcin (ucOC) (adjusted OR: 1.68, 95% CI: 1.02-2.78), and urinary pentocidine (adjusted OR: 2.51, 95% CI: 1.48-4.24) were associated with existing vertebral fracture. CONCLUSIONS: High frequent, multiple, and severe vertebral fractures were found in patients with RA compared to the controls. Low bone quality might be the cause of the frequent prevalence of vertebral fracture in patients with RA.

Transplantation of Scaffold-Free Cartilage-Like Cell-Sheets Made from Human Bone Marrow Mesenchymal Stem Cells for Cartilage Repair: A Preclinical Study.

OBJECTIVE: The object of this study was to determine culture conditions that create stable scaffold-free cartilage-like cell-sheets from human bone marrow-derived mesenchymal stem cells (hBMSCs) and to assess their effects after transplantation into osteochondral defects in nude rats. DESIGN: (Experiment 1) The hBMSCs were harvested from 3 males, the proliferative and chondrogenic capacities were assessed at passage 1, and the cells were expanded in 3 different culture conditions: (1) 5% fetal bovine serum (FBS), (2) 10% FBS, and (3) 5% FBS with fibroblast growth factor 2 (FGF-2). The cells were harvested and made chondrogenic pellet culture. The cell proliferation rate, glycosaminoglycan/DNA ratio, and safranin-O staining intensity of pellets cultured condition 3 were higher than those of conditions 1 and 2. (Experiment 2) The hBMSCs were expanded and passaged 3 times under culture condition 3, and fabricate the cell-sheets in chondrogenic medium either with or without FBS. The cell-sheets fabricated with FBS maintained their size with flat edges. (Experiment 3) The cell-sheets were transplanted into osteochondral defects in nude rats. Histological analysis was performed at 2, 4, and 12 weeks after surgery. RESULTS: The osteochondral repair was better after sheet transplantation than in the control group and significantly improved Wakitani score. Immunostaining with human-specific vimentin antibody showed that the transplanted cells became fewer and disappeared at 12 weeks. CONCLUSIONS: These results indicate that culture with FGF-2 may help to quickly generate sufficient numbers of cells to create stable and reliable scaffold-free cartilage-like cell-sheets, which contribute to the regeneration of osteochondral defects.

Levels of interleukin-1 beta can predict response to tocilizumab therapy in rheumatoid arthritis: the PETITE (predictors of effectiveness of tocilizumab therapy) study.

Predicting the responses of patients with rheumatoid arthritis (RA) to tocilizumab is difficult, because inflammatory markers such as C-reactive protein rapidly normalize regardless of clinical efficacy. We aimed to identify factors that could predict response to tocilizumab. Sixty-five patients completed 52 weeks of tocilizumab therapy. Serum fibrinogen, D-dimer and interleukin (IL)-1ß levels were measured at baseline and after 4 weeks of therapy. Clinical responses to tocilizumab were assessed using disease activity score 28-erythrocyte sedimentation rate and the clinical disease activity index at baseline and after 52 weeks of therapy (UMIN Clinical Trials Registry No. UMIN000002246). Mean age was 60.5 years (range 22-85 years). Mean disease duration was 11.2 years (range 0-45 years). All patients had moderate-to-severe disease activity and were resistant to disease-modifying anti-rheumatic drugs and/or other biologics. Baseline IL-1ß levels were significantly lower in responders than in non-responders (p = 0.045), but multiple logistic regression analysis found no significant difference (adjusted odds ratio 2.74; 95 % confidence interval 0.84-8.95; p = 0.096). Low D-dimer and IL-1ß levels at 4 weeks predicted greater decrease in disease activity after 52 weeks of treatment (p = 0.005 and p < 0.001, respectively). Effects of tocilizumab at 52 weeks could be predicted from D-dimer and IL-1ß levels after 4 weeks of tocilizumab treatment. These markers might be more useful than current inflammatory markers for early-stage prediction of response to tocilizumab in RA.

Articular cartilage endurance and resistance to osteoarthritic changes require transcription factor Erg.

OBJECTIVE: To determine whether and how the transcription factor Erg participates in the genesis, establishment, and maintenance of articular cartilage. METHODS: Floxed Erg mice were mated with Gdf5-Cre mice to generate conditional mutants lacking Erg in their joints. Joints of mutant and control mice were subjected to morphologic and molecular characterization and also to experimental surgically induced osteoarthritis (OA). Gene expression, promoter reporter assays, and gain- and loss-of-function in vitro tests were used to characterize molecular mechanisms of Erg action. RESULTS: Conditional Erg ablation did not elicit obvious changes in limb joint development and overall phenotype in juvenile mice. However, as mice aged, joints of mutant mice degenerated spontaneously and exhibited clear OA-like phenotypic defects. Joints in juvenile mutant mice were more sensitive to surgically induced OA and became defective sooner than operated joints in control mice. Global gene expression data and other studies identified parathyroid hormone-related protein (PTHrP) and lubricin as possible downstream effectors and mediators of Erg action in articular chondrocytes. Reporter assays using control and mutated promoter-enhancer constructs indicated that Erg acted on Ets DNA binding sites to stimulate PTHrP expression. Erg was up-regulated in severely affected areas in human OA articular cartilage but remained barely appreciable in areas of less affected cartilage. CONCLUSION: The study shows for the first time that Erg is a critical molecular regulator of the endurance of articular cartilage during postnatal life and that Erg can mitigate spontaneous and experimental OA. Erg appears to do this through regulating expression of PTHrP and lubricin, factors known for their protective roles in joints.

Effect of the direct injection of bone marrow mesenchymal stem cells in hyaluronic acid and bone marrow stimulation to treat chondral defects in the canine model.

INTRODUCTION: The purpose of this study was to assess the direct injection of bone marrow-derived mesenchymal stem cells (BMSCs) suspended in hyaluronic acid (HA) combined with drilling as a treatment for chondral defects in a canine model. METHODS: Tibial bone marrow was aspirated, and BMSCs were isolated and cultured. One 8.0-mm diameter chondral defect was created in the femoral groove, and nine 0.9-mm diameter holes were drilled into the defect. BMSCs (2.14 × 107 cells) suspended in HA were injected into the defect. HA alone was injected into a similar defect on the contralateral knee as a control. Animals were sacrificed at 3 and 6 months. RESULTS: Although the percentage of coverage assessed macroscopically was significantly better at 6 months than at 3 months in both the BMSC (p = 0.02) and control (p = 0.001) groups, there were no significant differences in the International Cartilage Repair Society grades. The Wakitani histological score was significantly better at 6 months than at 3 months in the BMSC and control groups. While the control defects were mostly filled with fibrocartilage, several of the defects in the BMSC group contained hyaline-like cartilage. The mean Wakitani scores of the BMSC group improved from 7.0 ± 1.0 at 3 months to 4.6 ± 0.9 at 6 months, and those of the control group improved from 9.4 ± 1.2 to 6.0 ± 0.6. The BMSC group showed significantly better regeneration than the control group at 3 months (p = 0.04), but the difference at 6 months was not significant (p = 0.06). CONCLUSIONS: The direct injection of BMSCs in HA combined with drilling enhanced cartilage regeneration.