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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.

Introduction: Mesenchymal stem/stromal cell (MSC)-based therapies in cartilage repair and osteoarthritis (OA) treatment have gained attention. Recently, there has been increasing evidence to suggest that MSCs secrete a wide range of trophic factors to modulate the injured tissue environment. Extracellular vesicles (EVs) have been suggested to explain the positive and sometimes curative effect, through the paracrine stimulation of the resident progenitor cells to aid in repair. Objectives: To give a narrative review of the current evidence for MSC-derived EV’s potential therapeutic effects and future perspectives for the treatment of OA. Methods: A literature search was conducted using the MEDLINE databases in November 2023. A total of 24 animal studies describing EV, including exosomes utilized in cartilage and OA treatments were identified and analyzed. Results: Preclinical animal studies indicated that EVs could enhance the therapeutic effects for the treatment of OA, although the purification methods for EVs and their cell sources would be highly relevant to therapeutic efficacy. In contrast, there have been no published clinical studies regarding EVs for the treatment of OA, and thus, properly controlled clinical trials and regulations are essential steps in future clinical applications. Conclusions: The current evidences suggest that the administration of MSC-derived EVs into damaged joints could effectively reduce cartilage loss and alleviate the progression of OA. In contrast, there are still several potential problems to be solved, including their classification, safety and toxicity in clinical use, as well as optimal dosage and frequency for human administration.

Abstract 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.

Abstract 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.

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.

Abstract Purpose Chondrocyte -based tissue engineering has been a promising option for the treatment of cartilage lesions. In previous literature, TD198946 has been shown to promote chondrogenic differentiation which could prove useful in cartilage regeneration therapies. Our study aimed to investigate the effects of TD198946 in generating engineered cartilage using dedifferentiated chondrocyte-seeded collagen scaffolds treated with TD198946. Methods Articular chondrocytes were isolated from mini pig knees and expanded in 2-dimensional cell culture and subsequently used in the experiments. 3-D pellets were then cultured for two weeks. Cells were also cultured in a type I collagen scaffolds for four weeks. Specimens were cultured with TD198946, BMP-2, or both in combination. Outcomes were determined by gene expression levels of RUNX1, SOX9, ACAN, COL1A1, COL2A1 and COL10A1, the glycosaminoglycan content, and characteristics of histology and immunohistochemistry. Furthermore, the maturity of the engineered cartilage cultured for two weeks was evaluated through subcutaneous implantation in nude mice for four weeks. Results Addition of TD198946 demonstrated the upregulation of gene expression level except for ACAN, type II collagen and glycosaminoglycan synthesis in both pellet and 3D scaffold cultures. TD198946 and BMP-2 combination cultures showed higher chondrogenic differentiation than TD198946 or BMP-2 alone. The engineered cartilage maintained its extracellular matrices for four weeks post implantation. In contrast, engineered cartilage treated with either TD198946 or BMP-2 alone was mostly absorbed. Conclusions Our results indicate that TD198946 could improve quality of engineered cartilage by redifferentiation of dedifferentiated chondrocytes pre-implantation and promoting collagen and glycosaminoglycan synthesis.

BackgroundIt 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.MethodsNine 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-β1. 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).ResultsThe 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.ConclusionIn 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 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.

Abstract Articular cartilage does not heal spontaneously due to its limited healing capacity, and thus effective treatments for cartilage injuries has remained challenging. Since the first report by Brittberg et al. in 1994, autologous chondrocyte implantation (ACI) has been introduced into the clinic. Recently, as an alternative for chondrocyte-based therapy, mesenchymal stem cell (MSC)-based therapy has received considerable research attention because of the relative ease in handling for tissue harvest, and subsequent cell expansion and differentiation. In this review, we discuss the latest developments regarding stem cell-based therapies for cartilage repair, with special focus on recent scaffold-free approaches.

Abstract Background In the meniscal repair procedures, a high ultimate load capacity and low cyclic creep at the repair site are favorable and lead to good biological incorporation of the tear site after surgery. Previous biomechanical tensile tests of the meniscal sutures have identified the suture knot as the weakest point. We hypothesized that the strength of a suture knot depends on the suture shape, and therefore, we compared three differently shaped suture materials composed of the same material and quantity per length. The purpose of this study was to determine whether a novel flat and wide repair material (FWRM), which consists of braided multi-threads that are cross-sectionally flat and wide, improves the ultimate load of knot breakage in a biomechanical experiment using a porcine trans-capsular meniscal repair model. Methods Eighteen fresh-frozen porcine knees (n = 6 in each group) were used. A longitudinal tear in the middle segment of the medial meniscus was created and repaired with a trans-capsular inside-out method using the following suture materials: No. 2–0 braided polyester conventional suture, hollow suture, and FWRM. After the separation of the inner segment of the meniscus with leaving, the suture stability of the repaired menisci was biomechanically analyzed with a video camera system for widening after a cyclic load between 5 and 20 N was applied 300 times. Ultimate failure load and stiffness at 5 mm/ min were also analyzed. Results We found no significant difference in suture widening after cyclic load tests [conventional suture, mean 0.51 mm (S.D. 0.39 mm); hollow suture, mean 0.23 mm (S.D. 0.11 mm); and FWRM, mean 0.54 mm (S.D. 0.08 mm)]. The failure mode in all specimens was knot breakage. Compared with those of the other groups, the ultimate failure load of FWRM was statistically significantly higher in the load-to-failure tests (conventional suture, mean 58.8 N [S.D. 8.25 N]; hollow suture, mean 79.4 N [S.D. 10.2 N]; and FWRM, mean 97.4 N [S.D. 3.65 N]; p

Vibration acceleration through whole body vibration has been reported to promote fracture healing. However, the mechanism responsible for this effect remains unclear. Purpose of this study was to determine whether vibration acceleration directly affects cells around the fracture site and promotes endochondral ossification. Four-week-old female Wistar Hannover rats were divided into two groups (vibration [V group] and control [C group]). The eighth ribs on both sides were cut vertically using scissors. From postoperative day 3 to 11, vibration acceleration using Power Plate® (30 Hz, low amplitude [30-Low], 10 min/day) was applied in the V group. Mature calluses appeared earlier in the V group than in the C group by histological analysis. The GAG content in the fracture callus on day 6 was significantly higher in the V group than in the C group. The mRNA expressions of SOX-9, aggrecan, and Col-II in the fracture callus on day 6 and Col-X on day 9 were significantly higher in the V group than in the C group. For in vitro analysis, four different conditions of vibration acceleration (30 or 50 Hz with low or high amplitude [30-Low, 30-High, 50-Low, and 50-High], 10 min/day) were applied to a prechondrogenic cell (ATDC5) and an undifferentiated cell (C3H10T1/2). There was no significant difference in cell proliferation between the control and any of the four vibration conditions for both cell lines. For both cell lines, alcian blue staining was greater under 30-Low and 50-Low conditions than under control as well as 30-High and 50-High conditions on days 7 and 14. Vibration acceleration under 30-L condition upregulated chondrogenic gene expressions of SOX-9, aggrecan, Col-II, and Col-X. Low-amplitude vibration acceleration can promote endochondral ossification in the fracture healing in vivo and chondrogenic differentiation in vitro.

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.

Meniscal injuries have posed a challenging problem for many years, especially considering that historically the meniscus was considered to be a structure with no important role in the knee joint. This led to earlier treatments aiming at the removal of the entire structure in a procedure known as a meniscectomy. However, with the current understanding of the function and roles of the meniscus, meniscectomy has been identified to accelerate joint degradation significantly and is no longer a preferred treatment option in meniscal tears. Current therapies are now focused to regenerate, repair, or replace the injured meniscus to restore its native function. Repairs have improved in technique and materials over time, with various implant devices being utilized and developed. More recently, strategies have applied stem cells, tissue engineering, and their combination to potentiate healing to achieve superior quality repair tissue and retard the joint degeneration associated with an injured or inadequately functioning meniscus. Accordingly, the purpose of this current review is to summarize the current available pre-clinical and clinical literature using stem cells and tissue engineering for meniscal repair and regeneration.

Mesenchymal stem cells (MSCs) derived from induced pluripotent stem cells (iPSCs) are a promising cell source for the repair of skeletal disorders. Recently, neural crest cells (NCCs) were reported to be effective for inducing mesenchymal progenitors, which have potential to differentiate into osteochondral lineages. Our aim was to investigate the feasibility of MSC-like cells originated from iPSCs via NCCs for osteochondral repair. Initially, MSC-like cells derived from iPSC-NCCs (iNCCs) were generated and characterized in vitro. These iNCC-derived MSC-like cells (iNCMSCs) exhibited a homogenous population and potential for osteochondral differentiation. No upregulation of pluripotent markers was detected during culture. Second, we implanted iNCMSC-derived tissue-engineered constructs into rat osteochondral defects without any preinduction for specific differentiation lineages. The implanted cells remained alive at the implanted site, whereas they failed to repair the defects, with only scarce development of osteochondral tissue in vivo. With regard to tumorigenesis, the implanted cells gradually disappeared and no malignant cells were detected throughout the 2-month follow-up. While this study did not show that iNCMSCs have efficacy for repair of osteochondral defects when implanted under undifferentiated conditions, iNCMSCs exhibited good chondrogenic potential in vitro under appropriate conditions. With further optimization, iNCMSCs may be a new source for tissue engineering of cartilage.

Mesenchymal stem cell- (MSC-) based therapy is a promising treatment for cartilage. However, repair tissue in general fails to regenerate an original hyaline-like tissue. In this study, we focused on increasing the expression levels for insulin-like growth factor-1 (IGF-1) to improve repair tissue quality. The IGF-1 gene was introduced into human synovial MSCs with a lentiviral vector and examined the levels of gene expression and morphological status of MSCs under chondrogenic differentiation condition using pellet cultures. The size of the pellets derived from IGF-1-MSCs were significantly larger than those of the control group. The abundance of glycosaminoglycan (GAG) was also significantly higher in the IGF-1-MSC group. The histology of the IGF-1-induced pellets demonstrated similarities to hyaline cartilage without exhibiting features of a hypertrophic chondrocyte phenotype. Expression levels for the Col2A1 gene and protein were significantly higher in the IGF-1 pellets than in the control pellets, but expression levels for Col10, MMP-13, ALP, and Osterix were not higher. Thus, IGF-1 gene transfer to human synovial MSCs led to an improved chondrogenic differentiation capacity without the detectable induction of a hypertrophic or osteogenic phenotype.

We have been developing a new tissue engineering technique for cartilage repair using a scaffold-free tissue engineered construct (TEC) bio-synthesized from synovium-derived mesenchymal stem cells (MSCs). A round-shaped chondral defect of 8.5 mm in diameter and 2.0 mm in depth created on the medial condyle of immature (4-month-old) and mature (12-month-old) porcine femur was filled with the TEC. Six months after surgery, a cylindrically shaped specimen of 4 mm in diameter and 4-5 mm in depth was extracted. Stiffness measurements were carried out on the specimen using an AFM after the surface image of the specimen was obtained. The TEC-treated tissues exhibited more irregular surface as compared with normal cartilage, regardless of animal maturity. The stiffness of the superficial layer of the TEC-treated tissue was significantly lower than those of the normal cartilage, indicating 6.8±2.3 mN/m in the immature group and 8.8±2.3 mN/m in the mature group. Histological observation indicated that the defects were repaired with a hyaline cartilage-like tissue with positive Safranine O staining, regardless of animal maturity. However, the superficial layer of the repaired tissues was negatively stained with Safranine O. The present study suggests that the recovery of the superficial layer is delayed in both immature and mature animals although the treatment with TEC enhances the repair of partial chondral defects.

Meniscal injuries have posed a challenging problem for many years, especially considering that historically the meniscus was considered to be a structure with no important role in the knee joint. This led to earlier treatments aiming at the removal of the entire structure in a procedure known as a meniscectomy. However, with the current understanding of the function and roles of the meniscus, meniscectomy has been identified to accelerate joint degradation significantly and is no longer a preferred treatment option in meniscal tears. Current therapies are now focused to regenerate, repair, or replace the injured meniscus to restore its native function. Repairs have improved in technique and materials over time, with various implant devices being utilized and developed. More recently, strategies have applied stem cells, tissue engineering, and their combination to potentiate healing to achieve superior quality repair tissue and retard the joint degeneration associated with an injured or inadequately functioning meniscus. Accordingly, the purpose of this current review is to summarize the current available pre-clinical and clinical literature using stem cells and tissue engineering for meniscal repair and regeneration. [ABSTRACT FROM AUTHOR]