James H. Hui

Autologous Bone Marrow–Derived Mesenchymal Stem Cells Versus Autologous Chondrocyte Implantation An Observational Cohort Study

Background: First-generation autologous chondrocyte implantation has limitations, and introducing new effective cell sources can improve cartilage repair. Purpose: This study was conducted to compare the clinical outcomes of patients treated with first-generation autologous chondrocyte implantation to patients treated with autologous bone marrow–derived mesenchymal stem cells (BMSCs). Study Design: Cohort study; Level of evidence, 3. Methods: Seventy-two matched (lesion site and age) patients underwent cartilage repair using chondrocytes (n = 36) or BMSCs (n = 36). Clinical outcomes were measured before operation and 3, 6, 9, 12, 18, and 24 months after operation using the International Cartilage Repair Society (ICRS) Cartilage Injury Evaluation Package, which included questions from the Short-Form Health Survey, International Knee Documentation Committee (IKDC) subjective knee evaluation form, Lysholm knee scale, and Tegner activity level scale. Results: There was significant improvement in the patients’ quality of life (physical and mental components of the Short Form-36 questionnaire included in the ICRS package) after cartilage repair in both groups (autologous chondrocyte implantation and BMSCs). However, there was no difference between the BMSC and the autologous chondrocyte implantation group in terms of clinical outcomes except for Physical Role Functioning, with a greater improvement over time in the BMSC group (P = .044 for interaction effect). The IKDC subjective knee evaluation (P = .861), Lysholm (P = .627), and Tegner (P = .200) scores did not show any significant difference between groups over time. However, in general, men showed significantly better improvements than women. Patients younger than 45 years of age scored significantly better than patients older than 45 years in the autologous chondrocyte implantation group, but age did not make a difference in outcomes in the BMSC group. Conclusion: Using BMSCs in cartilage repair is as effective as chondrocytes for articular cartilage repair. In addition, it required 1 less knee surgery, reduced costs, and minimized donor-site morbidity.

Identification of Common Pathways Mediating Differentiation of Bone Marrow‐ and Adipose Tissue‐Derived Human Mesenchymal Stem Cells into Three Mesenchymal Lineages

Mesenchymal stem cells derived from human bone marrow (hBMSCs) and human adipose tissue (hAMSCs) represent a useful source of progenitor cells for cell therapy and tissue engineering. However, it is not clear what the similarities and differences between them are. Like hBMSCs, hAMSCs can differentiate into osteogenic, adipogenic, and chondrogenic cells. Whether MSCs derived from different tissue sources represent fundamentally similar or different cell types is not clear. Given the possible different sources of MSCs for cell therapy, a comprehensive comparison of the different MSCs would be very useful. Here, we compared the transcriptome profile of hAMCS and hBMSCs during directed differentiation into bone, cartilage, and fat. Our data revealed considerable similarities between bone marrow‐derived MSCs (BMSCs) and adipose tissue‐derived MSCs (AMSCs). We uncovered an interesting bifurcation of pathways in both BMSCs and AMSCs, in which osteogenesis and adipogenesis appear to be linked in a differentiation branch separate from chondrogenesis. Our data suggest that although a set of common genes may be needed for early differentiation into all three lineages, a different set of signature genes is associated with maturation into fully differentiated cells. The recruitment of different late differentiation factors explains and supports our conclusion that BMSCs differentiate more efficiently into bone and cartilage, whereas AMSCs differentiate better into adipocytes. This study not only generated a rich database for continuing molecular characterization of various MSCs but also provided a rational basis for assessing qualities of MSCs from different sources for the purpose of cell‐based therapy and tissue engineering.

Bone marrow-derived mesenchymal stem cells influence early tendon-healing in a rabbit achilles tendon model.

BACKGROUND A repaired tendon needs to be protected for weeks until it has accrued enough strength to handle physiological loads. Tissue-engineering techniques have shown promise in the treatment of tendon and ligament defects. The present study tested the hypothesis that bone marrow-derived mesenchymal stem cells can accelerate tendon-healing after primary repair of a tendon injury in a rabbit model. METHODS Fifty-seven New Zealand White rabbits were used as the experimental animals, and seven others were used as the source of bone marrow-derived mesenchymal stem cells. The injury model was a sharp complete transection through the midsubstance of the Achilles tendon. The transected tendon was immediately repaired with use of a modified Kessler suture and a running epitendinous suture. Both limbs were used, and each side was randomized to receive either bone marrow-derived mesenchymal stem cells in a fibrin carrier or fibrin carrier alone (control). Postoperatively, the rabbits were not immobilized. Specimens were harvested at one, three, six, and twelve weeks for analysis, which included evaluation of gross morphology (sixty-two specimens), cell tracing (twelve specimens), histological assessment (forty specimens), immunohistochemistry studies (thirty specimens), morphometric analysis (forty specimens), and mechanical testing (sixty-two specimens). RESULTS There were no differences between the two groups with regard to the gross morphology of the tendons. The fibrin had degraded by three weeks. Cell tracing showed that labeled bone marrow-derived mesenchymal stem cells remained viable and present in the intratendinous region for at least six weeks, becoming more diffuse at later time-periods. At three weeks, collagen fibers appeared more organized and there were better morphometric nuclear parameters in the treatment group (p < 0.05). At six and twelve weeks, there were no differences between the groups with regard to morphometric nuclear parameters. Biomechanical testing showed improved modulus in the treatment group as compared with the control group at three weeks (p < 0.05) but not at subsequent time-periods. CONCLUSIONS Intratendinous cell therapy with bone marrow-derived mesenchymal stem cells following primary tendon repair can improve histological and biomechanical parameters in the early stages of tendon-healing.

Injectable mesenchymal stem cell therapy for large cartilage defects--a porcine model.

Current techniques in biological resurfacing of cartilage defects require an open arthrotomy or arthroscopy and involve the direct transplantation of isolated cells and/or scaffolds or whole tissue grafts with chondrogenic potential onto the cartilage defect. Our study investigates the possibility of direct intra‐articular injection of mesenchymal stem cells suspended in hyaluronic acid (HA) as an alternative to the much more invasive methods currently available. A partial‐thickness (without penetration of the subchondral bone) cartilage defect was created in the medial femoral condyle of an adult minipig. Mesenchymal stem cells from the iliac crest marrow of the same pig harvested in a separate procedure and suspended in 2 milliliters of hylan G‐F 20 (Synvisc) were injected intra‐articularly after the creation of the defect. This was followed by two more injections of hylan G‐F 20 (HA) at weekly intervals. Either saline or HA was injected into the knees of the controls. The pigs were sacrificed at 6 and 12 weeks for morphological and histological analysis. The cell‐treated groups showed improved cartilage healing both histologically and morphologically at 6 and 12 weeks compared with both controls. The use of intra‐articular injections of mesenchymal stem cells suspended in HA is a viable option for treating large cartilage defects. This would be further explored in clinical trials.

Injectable cultured bone marrow-derived mesenchymal stem cells in varus knees with cartilage defects undergoing high tibial osteotomy: a prospective, randomized controlled clinical trial with 2 years' follow-up.

PURPOSE To analyze the results of the use of intra-articular cultured autologous bone marrow-derived mesenchymal stem cell (MSC) injections in conjunction with microfracture and medial opening-wedge high tibial osteotomy (HTO). METHODS Fifty-six knees in 56 patients with unicompartmental osteoarthritic knees and genu varum were randomly allocated to the cell-recipient group (n = 28) or control group (n = 28). Patients who had a joint line congruity angle of more than 2°, malalignment of the knee from femoral causes, a fixed flexion deformity, or age older than 55 years were excluded. All patients underwent HTO and microfracture. The cell-recipient group received intra-articular injection of cultured MSCs with hyaluronic acid 3 weeks after surgery, whereas the control group only received hyaluronic acid. The primary outcome measure was the International Knee Documentation Committee (IKDC) score at intervals of 6 months, 1 year, and 2 years postoperatively. Secondary outcome measures were Tegner and Lysholm clinical scores and 1-year postoperative Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) scores. RESULTS The median age of the patients was 51 years, with a mean body mass index of 23.85. Both treatment arms achieved improvements in Tegner, Lysholm, and IKDC scores. After adjustment for age, baseline scores, and time of evaluation, the cell-recipient group showed significantly better scores. The effect of treatment showed an added improvement of 7.65 (95% confidence interval [CI], 3.04 to 12.26; P = .001) for IKDC scores, 7.61 (95% CI, 1.44 to 13.79; P = .016) for Lysholm scores, and 0.64 (95% CI, 0.10 to 1.19; P = .021) for Tegner scores. Magnetic resonance imaging scans performed 1 year after surgical intervention showed significantly better MOCART scores for the cell-recipient group. The age-adjusted mean difference in MOCART score was 19.6 (95% CI, 10.5 to 28.6; P < .001). CONCLUSIONS Intra-articular injection of cultured MSCs is effective in improving both short-term clinical and MOCART outcomes in patients undergoing HTO and microfracture for varus knees with cartilage defects. LEVEL OF EVIDENCE Level II, randomized controlled trial.

An Analysis of Soft Tissue Allograft Anterior Cruciate Ligament Reconstruction in a Rabbit Model A Short-Term Study of the Use of Mesenchymal Stem Cells to Enhance Tendon Osteointegration

Background Soft tissue allografts are essential for revision and multiple ligament surgeries in the knee, where donor-site morbidity is an issue. However, the use of allografts is associated with a higher failure rate of osteointegration. Mesenchymal stem cells (MSCs) are investigated as potential agents to enhance bone tunnel and tendon healing. Purpose This study was conducted to analyze the effect of coating allografts with MSCs on the quality and rate of osteointegration at the allograft tendon and bone interface, and the biomechanical properties of these enhanced anterior cruciate ligament (ACL) grafts compared with controls. Study Design Descriptive laboratory study. Methods Bilateral ACL reconstructions using Achilles tendon allografts were performed in 36 rabbits. On 1 limb, the graft was coated with autogenous MSCs in a fibrin glue carrier, while the contralateral limb served as a control with no MSCs. The reconstructions were assessed histologically and biomechanically at 2, 4, and 8 weeks. Results At 8 weeks, histologic analysis of the controls revealed the development of mature scar tissue resembling Sharpey fibers spanning the tendon-bone interface. In contrast, the MSC-enhanced reconstructions showed a mature zone of fibrocartilage blending from bone to the allograft, strongly resembling a normal ACL insertion. On biomechanical testing, the MSC-enhanced grafts had significantly higher load-to-failure rates than controls. However, the stiffness and Youngs modulus were lower in the treatment group. Conclusions The application of MSCs at the allograft tendon-bone interface during ACL reconstruction results in the development of an intervening zone of fibrocartilage. The use of MSCs to enhance allograft osteointegration is a novel method offering the potential of more physiologic and earlier healing, although further investigation must be conducted to improve the biomechanical strength. Clinical Relevance Mesenchymal stem cells can improve the biologic properties of soft tissue allograft healing. Combined with the decrease in donor-site morbidity, allografts are a viable choice for the sports medicine surgeon.

Evidence-Based Status of Microfracture Technique: A Systematic Review of Level I and II Studies.

PURPOSE Although many newer cartilage repair techniques have evolved over the past 2 decades, microfracture is still being advocated as the first line of treatment. Therefore it is timely to conduct a comprehensive review of the literature to assess and report on the current status of Level I and II evidence studies related to microfracture techniques. METHODS A literature search was carried out for Level I and II evidence studies on cartilage repair using the PubMed database. All the studies that dealt with microfracture techniques were selected. RESULTS Fifteen studies that involved microfracture techniques met the inclusion criteria of this review article, with 6 long-term and 9 short-term studies. These studies compared the clinical outcomes of microfracture with those of other treatments such as autologous chondrocyte implantation and osteochondral cylinder transfers. The majority of the studies reported poor clinical outcomes, whereas 2 studies reported the absence of any significant difference in the results. Small-sized lesions and younger patients showed good results in the short-term. However, osteoarthritis and treatment failures were observed at later postoperative periods of 5 to 10 years. CONCLUSIONS The use of microfracture for the treatment of small lesions in patients with low postoperative demands was observed to result in good clinical outcomes at short-term follow-up. Beyond 5 years postoperatively, treatment failure after microfracture could be expected regardless of lesion size. Younger patients showed better clinical outcomes. LEVEL OF EVIDENCE Level II, systematic review of Level I and II studies.

Biomimetic hydrogels for chondrogenic differentiation of human mesenchymal stem cells to neocartilage

In this study, a collagen mimetic peptide (CMP) containing a GFOGER sequence flanked by GPO repeat units (sequence: (GPO)(4)GFOGER(GPO)(4)GCG, CMP) was synthesized and chemically incorporated into a poly(ethylene glycol) (PEG) hydrogel through Michael addition chemistry. The PEG/collagen mimetic peptide hybrid hydrogel was used as a scaffold for encapsulation, proliferation and differentiation of human mesenchymal stem cells (hMSCs) into neocartilage/chondrocytes. Biophysical studies indicated that this peptide adopts stable triple helical conformation under simulated physiological conditions. Tetra hydroxyl PEG was functionalized to generate an acrylate group and reacted with the peptide, and hydrogels were formed in situ with the addition of cells and tetra sulfhydryl PEG via Michael addition. The effect of CMP on proliferation and chondrogenesis of hMSCs was investigated. The results demonstrated that PEG-CMP hydrogels provided a natural environment, which promoted chondrogenesis of hMSCs and enhanced secretion of cartilage specific ECM as compared to PEG hydrogels without the peptide. This was attributed to enhanced cell/matrix interactions via integrin beta1/GFOGER interactions. Further, chondrogenesis was found to be affected by matrix elasticity. Soft matrix induced a greater degree of chondrogenic differentiation; however, stiff matrix had an opposite effect, inhibiting chondrogenic differentiation probably due to limited mass transport. This soft PEG/CMP hydrogel shows promise as a biomimetic scaffold that provides a desirable environment for the chondrogenic differentiation of hMSCs and is useful for the repair of cartilage defects.

Effects of ectopic Nanog and Oct4 overexpression on mesenchymal stem cells.

Mesenchymal stem cells (MSCs) represent a source of pluripotent cells that are already in various phases of clinical application. However, the use of MSCs in tissue engineering has been hampered largely due to their limitations, including low proliferation, finite life span, and gradual loss of their stem cell properties during ex vivo expansion. Nanog and Oct4 are key transcription factors essential to the pluripotent and self-renewing phenotypes of undifferentiated embryonic stem cells (ESCs). To determine whether Nanog and Oct4 improve human bone marrow-MSC quality, we therefore established stable Nanog and Oct4 overexpressing MSCs using a lentiviral system and showed that this promoted cell proliferation and enhanced colony formation of MSCs. In differentiating MSCs, Nanog, and Oct4, overexpression had converse effects on adipogenesis of MSCs and Nanog overexpression slowed down adipogenesis, whereas Oct4 overexpression improved adipogenesis. Nanog and Oct4 overexpression both improved chondrogenesis. Microarray data showed many differences in transcriptional targets in undifferentiated MSCs overexpressing Nanog and Oct4. These results provide insight into the improvement of the stemness of MSCs by genetic modification with stemness-related genes.

Hyaluronic acid-based hydrogels functionalized with heparin that support controlled release of bioactive BMP-2.

Bone morphogenetic protein-2 (BMP-2) is a potent osteoinductive factor, yet its clinical use is limited by a short biological half-life, rapid local clearance and propensity for side effects. Heparin (HP), a highly sulfated glycosaminoglycan (GAG) that avidly binds BMP-2, has inherent biological properties that may circumvent these limitations. Here, we compared hyaluronan-based hydrogels formulated to include heparin (Heprasil™) with similar gels without heparin (Glycosil™) for their ability to deliver bioactive BMP-2 in vitro and in vivo. The osteogenic activity of BMP-2 released from the hydrogels was evaluated by monitoring alkaline phosphatase (ALP) activity and SMAD 1/5/8 phosphorylation in mesenchymal precursor cells. The osteoinductive ability of these hydrogels was determined in a rat ectopic bone model by 2D radiography, 3D μ-CT and histological analyses at 8 weeks post-implantation. Both hydrogels sustain the release of BMP-2. Importantly, the inclusion of a small amount of heparin (0.3% w/w) attenuated release of BMP-2 and sustained its osteogenic activity for up to 28 days. In contrast, hydrogels lacking heparin released more BMP-2 initially but were unable to maintain BMP-2 activity at later time points. Ectopic bone-forming assays using transplanted hydrogels emphasized the therapeutic importance of the initial burst of BMP-2 rather than its long-term osteogenic activity. Thus, tuning the burst release phase of BMP-2 from hydrogels may be advantageous for optimal bone formation.