A novel scaffold-free mesenchymal stem cell-derived tissue engineered construct for articular cartilage restoration - From basic to clinic.

Treatments for articular cartilage injuries are still challenging, due in part to its avascular and aneural surroundings. Since the first report of autologous chondrocyte implantation, cell-based therapies have been extensively studied with a variety of cell sources, including chondrocytes and mesenchymal stem/stromal cells (MSCs). Recently, MSC-based therapy has received considerable research attention because of the relative ease in handling for tissue harvest, and subsequent cell expansion and differentiation. Using such cells, we have originally developed a 3-dimensional scaffold-free tissue-engineered construct (TEC) through simple-cell culture methods and demonstrated its feasibility for cartilage repair and regeneration in the first-in-human clinical trial. This review summarizes our novel scaffold-free approaches to use MSC for the restoration of damaged articular cartilage, documenting the progression from basic to clinical studies.

Biologic therapies in stress fractures: Current concepts.

Stress fractures, a common overuse injury in physically active individuals, present a significant challenge for athletes and military personnel. Patients who sustain stress fractures have demanding training regimes where periods of rest and immobilisation have unacceptable negative consequences on sports goals and finances. Aside from being an overuse injury, there are various contributing risk factors that put certain individuals at risk of a stress fracture. The main two being nutritional deficiencies and hormonal variations, which have significant effects on bone metabolism and turnover. Historically, treatment of stress fractures focused on conservative strategies such as rest and immobilisation. Calcium and vitamin D deficiencies have been closely linked to stress fractures and so over time supplementation has also played a role in treatment. With the introduction of biologics into orthopaedics, newer treatment strategies have been applied to accelerate fracture healing and perhaps improve fracture callus quality. If such therapies can reduce time spent away from sport and activity, it would be ideal for treating stress fractures. This article aims to offer insights into the evolving landscape of stress fracture management. It investigates the pre-clinical evidence and available published clinical applications. Though fracture healing is well understood, the role of biologics for fracture healing is still indeterminate. Available literature for the use of biologic therapies in stress fractures are restricted and most reports have used biologics as a supplement to surgical fixation in subjects in studies that lack control groups. Randomised control trials have been proposed and registered by a few groups, with results awaited. Assessing individuals for risk factors, addressing hormonal imbalances and nutritional deficiencies seems like an effective approach to addressing the burden of stress fractures. We await better designed trials and studies to accurately determine the clinical benefit of adding biologics to the management of these injuries.

Repair of osteochondral defects: efficacy of a tissue-engineered hybrid implant containing both human MSC and human iPSC-cartilaginous particles.

Both mesenchymal stromal cells (MSC) and induced pluripotent stem cells (iPSC) offer the potential for repair of damaged connective tissues. The use of hybrid implants containing both human MSC and iPSC was investigated to assess their combined potential to yield enhanced repair of osteochondral defects. Human iPSC-CP wrapped with tissue engineered constructs (TEC) containing human MSC attained secure defect filling with good integration to adjacent tissue in a rat osteochondral injury model. The presence of living MSC in the hybrid implants was required for effective biphasic osteochondral repair. Thus, the TEC component of such hybrid implants serves several critical functions including, adhesion to the defect site via the matrix and facilitation of the repair via live MSC, as well as enhanced angiogenesis and neovascularization. Based on these encouraging studies, such hybrid implants may offer an effective future intervention for repair of complex osteochondral defects.

Five-Year Outcomes After Implantation of a Scaffold-Free Tissue-Engineered Construct Generated From Autologous Synovial Mesenchymal Stromal Cells for Repair of Knee Chondral Lesions.

Background: In an earlier study, a scaffold-free tissue-engineered construct (TEC) derived from autologous synovial membrane mesenchymal stromal cells (MSCs) was developed and demonstrated to be safe and effective for cartilage repair at 2 years postoperatively. Purpose: To investigate clinical outcomes and magnetic resonance imaging (MRI) findings at 5 years after implantation. Study Design: Case series; Level of evidence, 4. Methods: This was an observational first-in-human study limited to 5 patients (age, 28-46 years) with symptomatic knee chondral lesions (size, 1.5-3.0 cm2) on the medial femoral condyle, lateral femoral condyle, or femoral groove. Synovial MSCs were isolated from arthroscopic biopsy specimens and cultured to develop a TEC that matched the lesion size. The TECs were then implanted into chondral defects without fixation and assessed at up to 5 years postoperatively. The patients were clinically evaluated using the visual analog scale for pain, Lysholm score, Tegner score, and Knee injury and Osteoarthritis Outcome Score. An MRI scan evaluation was also performed for morphologic and compositional quality of the repair tissue at both 2 and 5 years of follow-up. Results: All clinical scores were significantly improved from the preoperative evaluation to the 2- and 5-year follow-ups and the results were stable over time. The MRI scan evaluation showed cartilage defects filled with newly generated tissues with good tissue integration to adjacent host cartilage over time. The cartilage thickness and surface smoothness of the repair cartilage were maintained up to 5 years postoperatively. The MOCART (magnetic resonance observation of cartilage repair tissue) 2.0 Knee Scores remained high at 5 years, although the total points decreased slightly. Conclusion: The results highlight the efficacy and feasibility of autologous scaffold-free TEC derived from synovial MSCs for regenerative cartilage repair via a sutureless and simple implantation procedure, showing good clinical outcomes and MRI findings with stable results at midterm follow-up. Further follow-up will be needed to assess the long-term quality of the repair tissue.

Freeze-dried noncoagulating platelet-derived factor concentrate is a safe and effective treatment for early knee osteoarthritis.

PURPOSE: While a wide variety of platelet-rich plasma (PRP) solutions has been developed, innovation continues. In this case, the freeze-dried platelet factor concentrate (PFC-FD) represents another step in PRP refinement. The preparation of PFC-FD at a central laboratory with freeze drying for shelf stabilization should provide additional quality improvements if clinical effectiveness can be demonstrated. Therefore, this study was undertaken to assess the safety and effectiveness of PFC-FD in a prospective open-label trial of patients suffering from knee osteoarthritis (OA). METHODS: 312 consecutive knee OA patients (67% female, mean age 63 ± 10 years), were prospectively recruited in an outpatient knee clinic in Japan. Of these, 10 (3.2%) were lost to follow-up at < 12 months and 17 (5.5%) sought additional knee therapy during the follow-up period. The primary outcome of interest was achievement of the OMERACT-OARSI responder criteria with secondary outcomes of adverse events and PROMs scores 1, 3, 6, 12 months following a single PFC-FD injection. RESULTS: 285 patients (91%) completed 12 month PROMs. The 17 who sought additional therapy were considered failures leaving an effective sample size of 302 for our primary outcome in which 62% of patients achieved OMERACT-OARSI responder status by 12 months. This varied by OA class with Kellgren-Lawrence grade 4 patients 3.6 times less likely to be responders than grade 1-2 patients. 6% of patients experienced a non-serious adverse event, primarily pain or swelling at the injection site. CONCLUSIONS: PFC-FD provides an observable clinical improvement in 62% of knee OA patients at 12 months post-injection with very little risk of any clinically relevant adverse event. Of course, nearly 40% of patients did not experience an observable clinical improvement, primarily among those with worse KL grades. LEVEL OF EVIDENCE: Therapeutic, Level II.

Small extracellular vesicles derived from human adipose-derived mesenchymal stromal cells cultured in a new chemically-defined contaminate-free media exhibit enhanced biological and therapeutic effects on human chondrocytes in vitro and in a mouse osteoarthritis model.

Human small extracellular vesicles (sEVs) derived from adipose-derived mesenchymal stromal cells (ASC) have been reported to suppress the progression of osteoarthritis (OA) in animal studies and subsequently, translation of this potential to assess their clinical efficacy is anticipated. However, fabrication protocols for sEVs to eliminate potential contamination by culture medium-derived components need to be established prior to their clinical use. The purpose of the present studies was to elucidate the influence of medium-derived contaminants on the biological effects of sEVs, and to establish isolation methods for sEVs using a new clinical grade chemically-defined media (CDM). The quantity and purity of ASC-derived sEVs cultured in four different CDMs (CDM1, 2, 3 and 4) were evaluated. The concentrates of the four media incubated without cells were used as the background (BG) control for each set of sEVs. The biological effect of sEVs fabricated in the four different CDMs on normal human articular chondrocytes (hACs) were evaluated in vitro using a variety of methodological assessments. Finally, the sEVs with the highest purity were tested for their ability to suppress the progression of knee OA mouse model. Analysis of the BG controls revealed that CDM1-3 contained detectable particles, while there was no visible contamination of culture media-derived components detected with CDM4. Accordingly, the sEVs fabricated with CDM4 (CDM4-sEVs) exhibited the highest purity and yield. Notably, the CDM4-sEVs were the most efficient in promoting the cellular proliferation, migration, chondrogenic differentiation, and anti-apoptotic activity of hACs. Furthermore, CDM4-sEVs significantly suppressed the osteochondral degeneration in vivo model. Small EVs derived from ASCs cultured in a CDM without detectable contaminants demonstrated enhanced biological effects on hACs and the progression of OA. Thus, sEVs isolated with CDM4 most optimally meet the requirements of efficacy and safety for assessment in their future clinical applications.

Two Uncommon Complications Related to Suture Knots After Meniscal All-Inside Suture Repair: A Case Report.

CASE: A 17-year-old male patient suffered a radial lateral meniscus tear and underwent an arthroscopic all-inside suture repair. After 7 months, the patient experienced catching. Magnetic resonance imaging and computed tomography revealed an intra-articular loose body without calcification, which was removed surgically. The excised specimen was histopathologically confirmed to be a necrotic meniscus fragment with a suture knot. In addition, cartilage damage because of suspected impingement by a residual suture knot was observed. After removing the loose body and knot, the patient's symptoms were relieved, and he returned to sports. CONCLUSION: Suture knot-related complications should be considered while performing meniscal repairs.

Clinical Safety and Effectiveness of Adipose-Derived Stromal Cell vs Stromal Vascular Fraction Injection for Treatment of Knee Osteoarthritis: 2-Year Results of Parallel Single-Arm Trials.

BACKGROUND: There are currently no disease-modifying treatments available for knee osteoarthritis (OA), although cultured adipose-derived stromal cells (ASCs) have shown promise in experimental models. However, given the regulatory limits on the use of cultured cells in humans, previous trials have focused primarily on the stromal vascular fraction (SVF) intra-articular injection. Therefore, the therapeutic value of ASCs for knee OA remains unknown. PURPOSE: To study ASC versus SVF intra-articular injection in patients with Kellgren-Lawrence (KL) knee OA grades 2 to 4 in parallel single-arm trials. STUDY DESIGN: Cohort study; Level of evidence, 2. METHODS: A total of 80 patients were enrolled, with 42 (72 knees) receiving ASC intra-articular injection and 38 (69 knees) receiving SVF. Patient-reported outcome measures were assessed at 1, 3, 6, 12, and 24 months using the Knee injury and Osteoarthritis Outcome Score 5 (KOOS5) and pain visual analog scale (VAS). The percentages of patients achieving the minimal clinically important difference (MCID) and Patient Acceptable Symptom State (PASS) were also calculated. Per protocol, a subset of the ASC group received an ASC booster injection after 6 months. A repeated-measures analysis of variance compared results between treatment arms and by KL grade over time. RESULTS: Patient-reported outcome measures improved substantially after both treatments (P < .05 at all time points), with the ASC group more likely to achieve the MCID (50% vs 24%; P = .01) and PASS (45% vs 24%; P = .04) for the pain VAS and the MCID (43% vs 16%; P = .02) for the KOOS5 at 12 months, although not at 24 months. Knees treated with ASC for KL grade 2/3 OA had significantly superior outcomes compared with those with KL grade 4 OA for the KOOS5 (P = .01) and pain VAS (P = .03), but no such difference was observed in knees treated with SVF. Three patients receiving ASCs (7%; all KL grade 3) sought additional nonoperative treatment by 24 months versus 9 patients receiving SVF (24%; all KL grade 3) (P = .06). ASC booster injections conferred no additional benefit. Notably, patients in the ASC cohort reported more injection-site pain and swelling after the booster injection than after the initial injection (P < .01). CONCLUSION: This represents the first head-to-head comparison of ASCs and SVF for the treatment of knee OA in humans. ASC and SVF injections both substantially improved knee pain and function at all follow-up time points, although ASC injections demonstrated significantly better improvements with regard to the MCID and PASS for the pain VAS and the MCID for the KOOS5 at 12 months. There appears to be no benefit to a booster ASC injection after initial treatment. Given less donor-site morbidity and equivalent superior outcomes at 2 years, the use of ASCs over SVF in the treatment of knee OA may be warranted.

[Efficacy of a Dexamethasone Dose Escalation Regimen with a Cumulative Dose for Preventing Oxaliplatin Hypersensitivity Reactions].

Oxaliplatin is a platinum complex antineoplastic agent widely used for chemotherapy of colorectal cancer. However, one of its side effects is hypersensitivity reactions, the incidence of which increases with a cumulative dose, thereby posing a difficulty to continue oxaliplatin use. Our hospital changed the premedication of oxaliplatin in August 2009 and September 2012. We retrospectively investigated the usefulness of these premedication changes. The results showed no significant difference in the incidence of hypersensitivity between the control group(12.1%)and the group receiving H1 and H2-blockers (12.3%); however, the incidence of hypersensitivity was significantly reduced in the group receiving increased dexamethasone based on the number of courses(2.7%). Therefore, our regimen was found to be effective in preventing hypersensitivity reactions to oxaliplatin.

Posterior Ankle Impingement Caused by Hyaline-Like Cartilage Generation in Ballet Dancers-A Report of 2 Cases.

Posterior ankle impingement syndrome is mainly seen in ballet dancers and frequently associated with specific movements in ballet such as pointe and demi pointe in which the whole-body weight is applied to the maximally plantar flexed ankle. We performed arthroscopic debridement for 2 dedicated ballet dancers on the intervening soft tissue causing posterior ankle impingement syndrome (PAIS). In both cases, T2-weighted magnetic resonance imaging (MRI) revealed low-signal intensity of meniscus-like soft tissue without abnormal osseous findings, connecting from the posterior side of the talus to Kager's fat pad. To examine the intervening soft tissue in detail, we performed histological evaluation by hematoxylin and eosin staining, Safranin O fast green staining, and immunohistochemistry for type I collagen and type II collagen. Hematoxylin and eosin staining showed that there was cartilage-like tissue including chondrocyte-like cells in contact with fibrous tissue. The extracellular matrix in the cartilage zone was consistently stained by Safranin O staining and type II collagen without any staining with type I collagen. These findings suggested that the meniscus-like soft tissue appearing as low-signal intensity on MRI at the posterior side of talus included hyaline-like cartilage. To the extent of our knowledge, these were rare cases of hyaline-like cartilage generation causing PAIS in ballet dancers, which might be associated with ballet specific movements resulting in chondrogenesis.

Suture Slippage During Anterior Cruciate Ligament Graft Passage Is Significantly Lower Using a Krackow Suture.

PURPOSE: To compare the suture slippage on a hamstring tendon graft prepared with a modified finger-trap device (SPEEDTRAP) with one prepared with Krackow stitch during graft passage through the tibial tunnel in ACL reconstruction. METHODS: Thirty-eight patients underwent anatomic triple-bundle anterior cruciate ligament reconstruction with 2 femoral and 3 tibial tunnels. After semitendinosus tendon was cut in half to make 2 grafts, the free ends of the proximal membranous portion (posterolateral [PL] graft) were prepared together with 2 sutures: (1) one SPEEDTRAP and one Krackow stitch for 20 cases (group A) and (2) double Krackow stitches on both sides for 18 cases (group B). Then, the PL graft was dye-marked at the proximal suture of SPEEDTRAP in group A and Krackow suture in group B and was inserted into the joint via tibial tunnel ahead of the loop side. The distance between the mark on the graft and the proximal suture of SPEEDTRAP or Krackow stitch was measured under arthroscopy after graft fixation at femur. Slippage was defined as 1 mm and more of distance between the mark and the proximal suture. RESULTS: Slippage was observed in 16 cases for SPEEDTRAP and in 2 for Krackow suture in group A, whereas one case showed slippage in group B. The slippage distance was 4.0 ± 2.9 mm for SPEEDTRAP and 0.2 ± 0.5 mm for Krackow stitch in group A (P < .001), whereas it was 0.1 ± 0.2 mm for double Krackow stitch in group B, showing a significant difference from SPEEDTRAP suture (P < .001). CONCLUSIONS: At the time of PL graft passage through the tibial tunnel in anterior cruciate ligament reconstruction, there was significantly less slippage observed with the Krackow stitch compared with the SPEEDTRAP stitch. LEVEL OF EVIDENCE: Level IV, therapeutic case series.

Return to the original sport at only 3 months after an Achilles tendon rupture by a combination of intra-tissue injection of freeze-dried platelet-derived factor concentrate and excessively early rehabilitation after operative treatment in a male basketball player: A case report.

BACKGROUND: Achilles tendon rupture is one of the most common serious injuries in athletes. Various studies to accelerate the healing process of the Achilles tendon have been performed as it takes a longer time to repair the tissue compared to other tendons. Here, we report a case of an acute Achilles tendon rupture in a male basketball player treated by a combination of an intra-tissue injection of freeze-dried platelet-derived factor concentrate, which included a platelet-derived growth factor with an early rehabilitation protocol after the operative treatment to facilitate the biological healing of the injured tendon tissue. To the best of our knowledge, this case is the first instance that enabled the athlete to return to original sport activity at only 3-months after the injury. CASE REPORT: A 23-year-old male basketball player who belonged to a university basketball team sustained an Achilles tendon rupture during running in a training match. The remaining time period until the final tournament of the university league as a senior player was only 3 months. The patient received a combination of an intra-tissue injection of freeze-dried platelet-derived factor concentrate and early rehabilitation protocol after operative treatment. Surgery was performed 4 days after the injury and the early rehabilitation protocols were applied postoperatively. A freeze-dried platelet-derived factor concentrate was injected into the ruptured site of the Achilles tendon under ultrasound guide at 4 weeks postoperatively. The patient could return to play at the pre-injury level without any symptoms and disfunctions at 3 months after surgery. At two years postoperatively, the patient could play basketball without symptoms or rerupture. CONCLUSIONS: We reported a case of an Achilles tendon rupture which was treated by a combination of intra-tissue injection of freeze-dried platelet-derived factor concentrate and an early rehabilitation protocol after the operative treatment. The patient could return to play basketball at the pre-injury activity level at only 3-months after the injury, suggesting that the role of applying excessively early rehabilitation of mechanical loading could facilitate tendon tissue healing when combined with an intra-tissue injection of freeze-dried platelet-derived factor concentrate.

Perifosine enhances the potential antitumor effect of 5-fluorourasil and oxaliplatin in colon cancer cells harboring the PIK3CA mutation.

Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutation in colon cancer contributes to the poor prognosis of the disease and chemoresistance of tumors. New therapies are needed; however, the lack of knowledge of the mechanism of chemoresistance has hindered progress. In this study, we investigated the mechanism of the reduced sensitivity of colon cancer cells to 5-fluorouracil (5-FU) and oxaliplatin (L-OHP), and the effects of perifosine, an Akt inhibitor that enhances the cytotoxicity of 5-FU and L-OHP in colon cancer cells harboring the PIK3CA mutation. The use of 5-FU or L-OHP alone or in combination induced significant death of Caco-2 cells (PIK3CA wild type), but only weakly decreased the viability of DLD-1 and SW948 cells harboring the PIK3CA mutation. The use of 5-FU and L-OHP, either alone or in combination, strongly suppressed Akt activation, Survivin, Bcl-2, and Bcl-xL expression, and enhanced Puma, phospho-p53, and p53 expression in Caco-2 cells than in DLD-1 cells. In addition, perifosine enhanced the cytotoxicity of the 5-FU and L-OHP combination, inhibited Akt activation and the expression of Survivin, Bcl-2, and Bcl-xL, and increased the expression of Puma, phospho-p53, and p53 in DLD-1 cells. These results indicate that PIK3CA mutation contributes to reduced sensitivity to 5-FU and L-OHP via Akt activation in colon cancer cells. Perifosine increases the efficacy of 5-FU and L-OHP by suppressing Akt activation. Thus, the use of an Akt inhibitor in combination with 5-FU and L-OHP may be beneficial in colon cancer with cells harboring the PIK3CA mutation.

Rhosin Suppressed Tumor Cell Metastasis through Inhibition of Rho/YAP Pathway and Expression of RHAMM and CXCR4 in Melanoma and Breast Cancer Cells.

The high mortality rate of cancer is strongly correlated with the development of distant metastases at secondary sites. Although Rho GTPases, such as RhoA, RhoB, RhoC, and RhoE, promote tumor metastasis, the main roles of Rho GTPases remain unidentified. It is also unclear whether rhosin, a Rho inhibitor, acts by suppressing metastasis by a downstream inhibition of Rho. In this study, we investigated this mechanism of metastasis in highly metastatic melanoma and breast cancer cells, and the mechanism of inhibition of metastasis by rhosin. We found that rhosin suppressed the RhoA and RhoC activation, the nuclear localization of YAP, but did not affect ERK1/2, Akt, or NF-κB activation in the highly metastatic cell lines B16BL6 and 4T1. High expression of YAP was associated with poor overall and recurrence-free survival in patients with breast cancer or melanoma. Treatment with rhosin inhibited lung metastasis in vivo. Moreover, rhosin inhibited tumor cell adhesion to the extracellular matrix via suppression of RHAMM expression, and inhibited SDF-1-induced cell migration and invasion by decreasing CXCR4 expression in B16BL6 and 4T1 cells. These results suggest that the inhibition of RhoA/C-YAP pathway by rhosin could be an extremely useful therapeutic approach in patients with melanoma and breast cancer.

Histological Analysis of Cartilage Defects Repaired with an Autologous Human Stem Cell Construct 48 Weeks Postimplantation Reveals Structural Details Not Detected by T2-Mapping MRI.

OBJECTIVE: The aim of this study was to elucidate the efficacy of T2-mapping MRI and correlation with histology for the evaluation of tissue repair quality following the first-in-human implantation of an autologous tissue engineered construct. DESIGN: We directly compared the results of T2-mapping MRI of cartilage repair tissue with the histology of a biopsy specimen from the corresponding area at 48 weeks postoperatively in 5 patients who underwent the implantation of a scaffold-free tissue-engineered construct generated from autologous synovial mesenchymal stem cells to repair an isolated cartilage lesion. T2 values and histological scores were compared at each of 2 layers of equally divided halves of the repair tissue (upper and lower zones). RESULTS: Histology showed that the repair tissue in the upper zone was dominated by fibrous tissue and the ratio of hyaline-like matrix increased with the depth of the repair tissue. There were significant differences between upper and lower zones in histological scores. Conversely, there were no detectable statistically significant differences in T2 value detected among zones of the repair tissue, but zonal differences were detected in corresponding healthy cartilage. Accordingly, there were no correlations detected between histological scores and T2 values for each repair cartilage zone. CONCLUSION: Discrepancies in the findings between T2 mapping and histology suggest that T2 mapping was limited in ability to detect details in the architecture and composition of the repair cartilage.

Current hip cartilage regeneration/repair modalities: a scoping review of biologics and surgery.

PURPOSE: The rapidly growing and emerging nature of biologics have made indications for regenerative and reparative hip therapies ever changing, with at times only early-stage evidence for their use. The purpose of this study was to review and summarize the currently available data on the management of hip cartilage injuries and osteoarthritis. METHODS: A scoping review of the available scientific literature for hip biologics was performed, with available evidence for hyaluronic acid (HA), platelet rich plasma (PRP), stem/stromal cells, microfracture, mosaicplasty, osteochondral allograft, and cell-based therapies investigated. RESULTS: To date, there exist better guidelines and further consensus concerning knee joint biologic treatments than the hip due to a greater number of studies as well as the more recent emergence of hip preservation approaches. However, increasing evidence is available for the selective implementation of biologics on an individualized basis with attention to lesion size and location. CONCLUSION: Orthopedic surgeons are at an exciting crossroads in medicine, where hip biologic therapies are evolving and increasingly available. Timetested interventions such as arthroplasty have shown good results and still have a major role to play but newer, regenerative approaches have the potential to effectively delay or reduce the requirement for such invasive procedures.

Evidence that TD-198946 enhances the chondrogenic potential of human synovium-derived stem cells through the NOTCH3 signaling pathway.

Human synovium-derived stem cells (hSSCs) are an attractive source of cells for cartilage repair. At present, the quality of tissue and techniques used for cartilage regeneration have scope for improvement. A small compound, TD-198946, was reported to enhance chondrogenic induction from hSSCs; however, other applications of TD-198946, such as priming the cell potential of hSSCs, remain unknown. Our study aimed to examine the effect of TD-198946 pretreatment on hSSCs. HSSCs were cultured with or without TD-198946 for 7 days during expansion culture and then converted into a three-dimensional pellet culture supplemented with bone morphogenetic protein-2 (BMP2) and/or transforming growth factor beta-3 (TGFß3). Chondrogenesis in cultures was assessed based on the GAG content, histology, and expression levels of chondrogenic marker genes. Cell pellets derived from TD-198946-pretreated hSSCs showed enhanced chondrogenic potential when chondrogenesis was induced by both BMP2 and TGFß3. Moreover, cartilaginous tissue was efficiently generated from TD-198946-pretreated hSSCs using a combination of BMP2 and TGFß3. Microarray analysis revealed that NOTCH pathway-related genes and their target genes were significantly upregulated in TD-198946-treated hSSCs, although TD-198946 alone did not upregulate chondrogenesis related markers. The administration of the NOTCH signal inhibitor diminished the effect of TD-198946. Thus, TD-198946 enhances the chondrogenic potential of hSSCs via the NOTCH3 signaling pathway. This study is the first to demonstrate the gradual activation of NOTCH3 signaling during chondrogenesis in hSSCs. The priming of NOTCH3 using TD-198946 provides a novel insight regarding the regulation of the differentiation of hSSCs into chondrocytes.

Potential of Soluble Decellularized Extracellular Matrix for Musculoskeletal Tissue Engineering - Comparison of Various Mesenchymal Tissues.

BACKGROUND: It is well studied that preparations of decellularized extracellular matrix (ECM) obtained from mesenchymal tissues can function as biological scaffolds to regenerate injured musculoskeletal tissues. Previously, we reported that soluble decellularized ECMs derived from meniscal tissue demonstrated excellent biocompatibility and produced meniscal regenerate with native meniscal anatomy and biochemical characteristics. We therefore hypothesized that decellularized mesenchymal tissue ECMs from various mesenchymal tissues should exhibit tissue-specific bioactivity. The purpose of this study was to test this hypothesis using porcine tissues, for potential applications in musculoskeletal tissue engineering. METHODS: Nine types of porcine tissue, including cartilage, meniscus, ligament, tendon, muscle, synovium, fat pad, fat, and bone, were decellularized using established methods and solubilized. Although the current trend is to develop tissue specific decellularization protocols, we selected a simple standard protocol across all tissues using Triton X-100 and DNase/RNase after mincing to compare the outcome. The content of sulfated glycosaminoglycan (sGAG) and hydroxyproline were quantified to determine the biochemical composition of each tissue. Along with the concentration of several growth factors, known to be involved in tissue repair and/or maturation, including bFGF, IGF-1, VEGF, and TGF-ß1. The effect of soluble ECMs on cell differentiation was explored by combining them with 3D collagen scaffold culturing human synovium derived mesenchymal stem cells (hSMSCs). RESULTS: The decellularization of each tissue was performed and confirmed both histologically [hematoxylin and eosin (H&E) and 4',6-diamidino-2-phenylindole (DAPI) staining] and on the basis of dsDNA quantification. The content of hydroxyproline of each tissue was relatively unchanged during the decellularization process when comparing the native and decellularized tissue. Cartilage and meniscus exhibited a significant decrease in sGAG content. The content of hydroxyproline in meniscus-derived ECM was the highest when compared with other tissues, while sGAG content in cartilage was the highest. Interestingly, a tissue-specific composition of most of the growth factors was measured in each soluble decellularized ECM and specific differentiation potential was particularly evident in cartilage, ligament and bone derived ECMs. CONCLUSION: In this study, soluble decellularized ECMs exhibited differences based on their tissue of origin and the present results are important going forward in the field of musculoskeletal regeneration therapy.

Osteochondral Injury, Management and Tissue Engineering Approaches.

Osteochondral lesions (OL) are a common clinical problem for orthopedic surgeons worldwide and are associated with multiple clinical scenarios ranging from trauma to osteonecrosis. OL vary from chondral lesions in that they involve the subchondral bone and chondral surface, making their management more complex than an isolated chondral injury. Subchondral bone involvement allows for a natural healing response from the body as marrow elements are able to come into contact with the defect site. However, this repair is inadequate resulting in fibrous scar tissue. The second differentiating feature of OL is that damage to the subchondral bone has deleterious effects on the mechanical strength and nutritive capabilities to the chondral joint surface. The clinical solution must, therefore, address both the articular cartilage as well as the subchondral bone beneath it to restore and preserve joint health. Both cartilage and subchondral bone have distinctive functional requirements and therefore their physical and biological characteristics are very much dissimilar, yet they must work together as one unit for ideal joint functioning. In the past, the obvious solution was autologous graft transfer, where an osteochondral bone plug was harvested from a non-weight bearing portion of the joint and implanted into the defect site. Allografts have been utilized similarly to eliminate the donor site morbidity associated with autologous techniques and overall results have been good but both techniques have their drawbacks and limitations. Tissue engineering has thus been an attractive option to create multiphasic scaffolds and implants. Biphasic and triphasic implants have been under explored and have both a chondral and subchondral component with an interface between the two to deliver an implant which is biocompatible and emulates the osteochondral unit as a whole. It has been a challenge to develop such implants and many manufacturing techniques have been utilized to bring together two unalike materials and combine them with cellular therapies. We summarize the functions of the osteochondral unit and describe the currently available management techniques under study.