Effective Cartilage Regeneration Research Articles

Transforming growth factor-β1-loaded RADA-16 hydrogel scaffold for effective cartilage regeneration

Cartilage repair remains a major challenge in clinical trials. These current cartilage repair materials can not effectively promote chondrocyte generation, limiting their practical application in cartilage repair. In this work, we develop an implantable scaffold of RADA-16 peptide hydrogel incorporated with TGF-β1 to provide a microenvironment for stem cell-directed differentiation and chondrocyte adhesion growth. The longest release of growth factor TGF-β1 release can reach up to 600 h under physiological conditions. TGF-β1/RADA-16 hydrogel was demonstrated to be a lamellar porous structure. Based on the cell culture with hBMSCs, TGF-β1/RADA-16 hydrogel showed excellent ability to promote cell proliferation, directed differentiation into chondrocytes, and functional protein secretion. Within 14 days, 80% of hBMSCs were observed to be directed to differentiate into vigorous chondrocytes in the co-culture of TGF-β1/RADA-16 hydrogels with hBMSCs. Specifically, these newly generated chondrocytes can secrete and accumulate large amounts of collagen II within 28 days, which can effectively promote the formation of cartilage tissue. Finally, the exploration of RADA-16 hydrogel-based scaffolds incorporated with TGF-β1 bioactive species would further greatly promote the practical clinical trials of cartilage remediation, which might have excellent potential to promote cartilage regeneration in areas of cartilage damage.

Improved Results From Medium- and Long-Term Outcomes of Autogenous Osteoperiosteal Grafting for Large Cystic Lesions of the Talus

To investigate the medium and long-term follow-up outcomes of large cystic medial osteochondral lesions of the talus (OLTs) treated with autologous tibial osteoperiosteal grafts from the medial tibia and to explore the risk factors influencing the treatment outcomes. The retrospective study included 75 patients with large cystic medial OLTs who underwent autologous osteoperiosteal cylinder graft taken from the medial tibia between January 2004 and August 2018. They were assessed preoperatively and postoperatively using a visual analog scale, the Orthopedic Foot & Ankle Society Ankle-Hindfoot Scale (AOFAS), and short-form 36-item Short Form Health Survey score. Magnetic resonance observation of cartilage repair tissue, second-look arthroscopy, and biopsy were used to evaluate the grafting areas. After a follow-up period of 6.3 years, the virtual analog scale score decreased to 1.47 ± 1.32, and the AOFAS and 36-item Short Form Health Survey scores increased to 82.78 ± 11.65 and 83.26 ± 8.49, respectively, all of which showed significant improvement over preoperative scores (P < .001), and the average magnetic resonance observation of cartilage repair tissue score was 82.6 ± 8.4 (56.0-91.6). Eight patients underwent a second-look arthroscopic examination and were rated by the International Cartilage Repair Society scale; of them, 2 patients were rated grade Ⅰ, 4 were rated grade Ⅱ, and 2 were rated grade Ⅲ. Three patients underwent grafting area biopsy during the second-look arthroscopy, and the results showed that the grafting areas were rich in chondrocytes. Large OLTs (≥200 mm2) and obesity (body mass index ≥25) were responsible for the poor improvement of AOFAS score, according to multivariate Cox regression analysis. Autologous osteoperiosteal grafting was an effective treatment for large cystic medial OLTs, with effective cartilage regeneration in the grafted areas in the medium and long term. However, the large OLTs and obesity may reduce the treatment outcomes. Level IV, case series.

Fabrication of N‐methyl morpholine‐N‐oxide modified bacterial exopolysaccharide as natural scaffold toward effective cartilage regeneration in monosodium iodoacetate induced experimental osteoarthritis

AbstractA non‐pathogenic strain of Ochrobactrum pseudintermedium C1 produces potent surface‐active exopolysaccharides which exhibits a porous scaffold‐like architecture. Such a property is quite prospective in facilitating housing of cells for in vivo tissue regeneration. This surfactant exopolysaccharide has been fabricated for augmenting such regenerative properties. Different vital parameters of the fabricated EPS like surface morphology, swelling behavior, in vitro degradation study, cytotoxicity and biocompatibility have been evaluated which helped postulate the applicative profile of this natural scaffold. Further, Ochrobactrum‐borne fabricated EPS, when applied as a scaffold in monosodium iodoacetate induced experimental osteoarthritis in Wistar rat model, helped significant chondrogenesis without leaving any residual amount of scaffold. The functional restoration of knee joints, assessment of chondroitin sulfate in damaged cartilage, diminution of stress induced indicators like reactive oxygen metabolite and histopathological findings quite appreciably favored the objectives. Moreover, a single high dose exhibited considerable efficacy compared to repetitive targeted administration of the scaffold at regular intervals. Thus, this novel biological macromolecule offers a green and cost‐effective alternative to various commercially available synthetic scaffolds that promote tissue regeneration.

Adipose stem cell transplantation using adhesive protein-based viscous immiscible liquid for cartilage reconstruction

Articular cartilage (AC) is the smooth tissue that covers the end of bones and absorbs external impacts. AC can be damaged by various types of impacts. Thus, damaged AC can trigger the development of osteoarthritis (OA). With the limited intrinsic healing capacity of AC, cell transplantation has been recognized as a possible means of AC reconstitution. However, transplanted stem cells disappear from the transplantation site rapidly, abrogating the original intent of cell transplantation. In the present work, a viscous immiscible liquid-phase bioadhesive using bioengineered mussel adhesion protein (MAP) was applied for prolonged retention of transplanted stem cells in defective AC and retained stem cells in situ. Through electrostatic interactions with high-molecular-weight hyaluronic acid (HA), MAP was formulated into a viscous immiscible liquid phase complex coacervate (MAP-HA) with self-encapsulation of stem cells. MAP-HA exhibited excellent rheological and adhesive properties without cytotoxicity. Through in vivo rabbit OA model evaluation, we proved that immobilizing transplanted cells and preventing dispersion into the joint cavity by MAP-HA can increase the survival of transplanted cells and enhance their therapeutic effects. Therefore, stem cell-encapsulated biocompatible MAP-HA can be used as a cell-retaining bioadhesive scaffold in effective cartilage regeneration.

Peedanil Gold, Herbo-Mineral Formulation, Moderates Cytokine Levels and Attenuates Pathophysiology in Monosodium Iodoacetate Induced Osteoarthritis in SD Rat Model.

The inflammatory cartilaginous degeneration of the articular joints, mostly those of knee, hips and hands, is osteoarthritis (OA). The available treatment strategies for osteoarthritis are designed for pain relief, molecular targeting, cartilage regeneration and surgical intervention. However, meta-analysis of clinical trials has shown these strategies to be sub-optimal, thereby, eliciting a need for investigating alternative options. The herbo-mineral formulation, Peedanil Gold (PN-G) has been used against joint pains and inflammation. In the current study, anti-osteoarthritic effects of PN-G were investigated in rat model of OA, induced by intra-articular injection of monosodium-iodoacetate. PN-G treatment improved the clinical and Kellgren & Lawrence scores; and rescued the osteoarthritic rats from hyperalgesia and allodynia. Besides, PN-G treatment ameliorated joint inflammation and abrogated in vivo osteoarthritic pathology through effective cartilage regeneration, measured radiologically and histopathologically. PN-G also reduced the levels of interleukin-6 (IL-6) and interleukin-1 beta (IL-1β), in a dose dependent manner, in inflamed human macrophagic THP-1 cells, thereby, reaffirming its anti-inflammatory property at cytosafe concentrations. Ultra High performance liquid chromatography (UHPLC) revealed the presence of several analgesic and anti-inflammatory phytocompounds, like ellagic acid, guggulsterone E, guggulsterone Z, 5-(hydroxymethyl) furfural, corilagin, cinnamic acid, ferulic acid, gallic acid and protocatechuic acid in PN-G. In conclusion, this study has succinctly demonstrated that PN-G is capable of relieving the clinical symptoms of osteoarthritis, which is measurable through the established osteoarthritic serum biomarker, Cartilage Oligomeric Matrix Protein (COMP).

Engineered Cartilage from Human Chondrocytes with Homozygous Knockout of Cell Cycle Inhibitor p21.

Osteoarthritis (OA) is a highly prevalent disease with limited treatment options. The search for disease-modifying OA therapies would benefit from a more comprehensive knowledge of the genetic variants that contribute to chondrocyte dysfunction and the barriers to cartilage regeneration. One goal of this study was to establish a system for producing engineered cartilage tissue from genetically defined primary human chondrocytes through genome editing and single-cell expansion. This process was utilized to investigate the functional effect of biallelic knockout of the cell cycle inhibitor p21. The use of ribonucleoprotein (RNP) CRISPR/Cas9 complexes targeting two sites in the coding region of p21 resulted in a high frequency (16%) of colonies with homozygous p21 knockout. Chondrogenic pellet cultures from expanded chondrocytes with complete loss of p21 produced more glycosaminoglycans (GAG) and maintained a higher cell number. Single-cell-derived colonies retained the potential for robust matrix production after expansion, allowing for analysis of colony variability from the same population of targeted cells. The effect of enhanced cartilage matrix production in p21 knockout chondrocytes persisted when matrix production from individual colonies was analyzed. Chondrocytes had lower levels of p21 protein with further expansion, and the difference in GAG production with p21 knockout was strongest at early passages. These results support previous findings that implicate p21 as a barrier to cartilage matrix production and regenerative capacity. Furthermore, this work establishes the use of genome-edited human chondrocytes as a promising approach for engineered tissue models containing user-defined gene knockouts and other genetic variants for investigation of OA pathogenesis. Impact Statement This work provides two important advances to the field of tissue engineering. One is the demonstration that engineered cartilage tissue can be produced from genetically defined populations of primary human chondrocytes. While CRISPR/Cas-9 genome editing has been extensively used in cell lines that divide indefinitely, this work extends the technique to an engineered tissue model system to support investigation of genetic changes that affect cartilage production. A second contribution is the finding that chondrocytes with p21 knockout synthesized more cartilage matrix tissue than unedited controls. This supports the continued investigation of p21 as a potential barrier to effective cartilage regeneration.

Proliferation medium in three-dimensional culture of auricular chondrocytes promotes effective cartilage regeneration invivo.

IntroductionCartilage regeneration have been attracted attentions because of the poor ability of cartilage tissues to regenerate. Three-dimensional (3D) culture of chondrocytes is considered to be advantageous for cartilage regeneration. Although it is plausible that maturation of the constructs before transplantation positively affects the chondrogenesis, matured constructs after cultures for longer periods do not necessarily result in effective cartilage regeneration. In this study, we compared different types of culture media including growth factors which are clinically available. We prepared differentiation medium containing insulin-like growth factor-1 (IGF-1), proliferation medium containing fibroblast growth factor-2 (FGF-2) and insulin, and combination of them, and compared their efficacies on chondrogenesis when used in 3D culture of engineered cartilage constructs.MethodsCartilage constructs were fabricated by auricular chondrocytes and atelocollagen, and they were 3D-cultured with four types of media: control medium, differentiation medium, proliferation medium, and combination medium. After 3 weeks of culture, the constructs were analyzed for cell number, gene and protein expressions and mechanical properties. The constructs were also transplanted into nude mice. After 8 weeks, the degree of cartilage regeneration was evaluated. Constructs manufactured with canine auricular chondrocytes were subjected to autologous transplantation into beagles and examined for cartilage regeneration.ResultsDuring 3D culture, remarkably high gene expression of type II collagen was detected in the construct cultured with the differentiation medium whereas cell apoptosis were suppressed in the proliferation medium. When transplanted into nude mice, the constructs 3D-cultured in the proliferation medium produced abundant cartilage matrices. In autologous implantation model, the construct cultured in the proliferation medium again showed better chondrogenesis than those in other media.ConclusionsThe present study indicates that 3D culture with the proliferation medium maintains the cell viability to potentiate the subsequent cartilage regeneration. Here, we propose that not only differentiation but also high cell viability accompanied by proliferation factors should be taken into account to improve cartilage regeneration.

Decorin Regulates the Aggrecan Network Integrity and Biomechanical Functions of Cartilage Extracellular Matrix.

Joint biomechanical functions rely on the integrity of cartilage extracellular matrix. Understanding the molecular activities that govern cartilage matrix assembly is critical for developing effective cartilage regeneration strategies. This study elucidated the role of decorin, a small leucine-rich proteoglycan, in the structure and biomechanical functions of cartilage. In decorin-null cartilage, we discovered a substantial reduction of aggrecan content, the major proteoglycan of cartilage matrix, and mild changes in collagen fibril nanostructure. This loss of aggrecan resulted in significantly impaired biomechanical properties of cartilage, including decreased modulus, elevated hydraulic permeability, and reduced energy dissipation capabilities. At the cellular level, we found that decorin functions to increase the retention of aggrecan in the neo-matrix of chondrocytes, rather than to directly influence the biosynthesis of aggrecan. At the molecular level, we demonstrated that decorin significantly increases the adhesion between aggrecan and aggrecan molecules and between aggrecan molecules and collagen II fibrils. We hypothesize that decorin plays a crucial structural role in mediating the matrix integrity and biomechanical functions of cartilage by providing physical linkages to increase the adhesion and assembly of aggrecan molecules at the nanoscale.

Expression of microRNA-892B in human mesenchymal stem cells promotes cartilage regeneration in athymic nude rat osteochondral defect model

Purpose: Articular cartilage (AC) is poorly supplied by blood vessels, nerves, and the lymphatic system. Therefore, AC has a very limited capacity that can be repaired after an injury. Although several therapeutic measures have been developed to promote the healing of AC, no widely accepted treatment has been established. But transfection of a cocktail of genes into cells has recently been attractive to stem cell differentiation. Here, based on our previous in vitro studies, we confirm in vivo effectiveness of miR-892b for cartilage regeneration in the rat osteochondral defect model in vivo. Our strategy modulating miR-892b gene can suggest a new strategy for effective cartilage regeneration in MSC mediated tissue engineering fields. Methods: The knee joints of the male arthymic nude rat were exposed by a medial parapatellar incision, and the trochlear groove was severed due to the lateral obstruction of the patella. A 1.5 mm outer diameter trephine drill was employed to create osteochondral defects (1.5 × 3.0 mm) in the trochlear groove of the femur. The osteochondral defects was managed using one of the following methods : filling with HCF only (Group 1, n = 3), filling with TGF-â1-loaded HCF (Group 2, n = 3); and filling with lenti-control/hMSC-loaded HCF (Group 3, n = 3); and filling with lenti-control/hMSC and TGF-â1-loaded HCF (Group 4, n = 3); and filling with lenti-miR-892b/hMSC and TGF-â1-loaded HCF (Group 5, n = 3). An equal volume of thrombin solution was simultaneously injected with HCF in all groups. Each group consisted of 3 rats. After 8 weeks, the rats were sacrificed. Results: After genomic DNA PCR, a sequence correspond to pri-miR-892b was cloned into NheI-EcoRI sites of pCDH-copGFP lentiviral plasmid (Fig. 1a). As shown in Fig. 1b, lentiviral control and lenti-miR-892b transduced hMSCs were dually labeled with a far-red fluorescent dye (CellVeu) for in vivo visualization. After 8 weeks of post-surgery, this far-red fluorescent signals were only detected osteochondral defects of CellVeu labeled hMSCs transplanted groups (Fig. 1c). The ICRS macroscopic score was highest in group 5 (lenti-miR-892b-transduced hMSCs and TGF-β1: 11.0), Group 5 had significantly higher score than the other groups (Fig. 1d). Histological assessment showed that group 5 defects had good healing and covered with hyaline cartilage-like tissue. Although group 4 defects had complete healing with hyaline cartilage-like tissue, remodeling status in subchondral bone of the defects was irregular and incomplete. Defects of the rest group were mostly filled with fibrous tissue (Fig. 1f). According to the Wakitani Histological Grading Scale, group 5 was significantly lower than other group in surface, cell distribution, and subchondral bone (Fig. 1e). Conclusions: In conclusion, we previously found that miR-892b can enhance in vitro chondrogenesis of hMSCs. Here, we additionally evaluated its in vivo efficacy using rat osteochondral defect model. The macroscopic assessment of the regenerated cartilages demonstrated that the defect had become firm and smooth after 8 weeks in Groups 4, 5 while a visible defect was present in Group 1, 2 and 3. However, the reconstitution of subchondral bone was better in Group 5 than in Groups 4. The histological score was significantly better in Group 5 than in the other groups. These in vivo results confirmed that the expression of miR-892b in hMSC under TGF-β1 stinuli might promote the healing of osteochondral defects.

Cartilage tissue formation through assembly of microgels containing mesenchymal stem cells

Current clinical approaches to treat articular cartilage degeneration provide only a limited ability to regenerate tissue with long-term durability and functionality. In this application, injectable bulk hydrogels and microgels containing stem cells can provide a suitable environment for tissue regeneration. However insufficient cell–cell interactions, low differentiation efficiency and poor tissue adhesion hinder the formation of high-quality hyaline type cartilage. Here, we have designed a higher order tissue-like structure using injectable cell-laden microgels as the building blocks to achieve human bone marrow-derived mesenchymal stem cell (hBMSC) long-term maintenance and chondrogenesis. We have demonstrated that a 4-arm poly(ethylene glycol)-N-hydroxysuccinimide (NHS) crosslinker induces covalent bonding between the microgel building blocks as well as the surrounding tissue mimic. The crosslinking process assembles the microgels into a 3D construct and preserves the viability and cellular functions of the encapsulated hBMSCs. This assembled microgel construct encourages upregulation of chondrogenic markers in both gene and glycosaminoglycan (GAG) expression levels. In addition, the regenerated tissue in the assembled microgels stained positively with Alcian blue and Safranin O exhibiting unique hyaline-like cartilage features. Furthermore, the immunostaining showed a favourable distribution and significantly higher content of type II collagen in the assembled microgels when compared to both the bulk hydrogel and pellet cultures. Collectively, this tissue adhesive hBMSC-laden microgel construct provides potential clinical opportunities for articular cartilage repair and other applications in regenerative medicine. Statement of SignificanceA reliable approach to reconstruct durable and fully functional articular cartilage tissue is required for effective clinical therapies. Here, injectable hydrogels together with cell-based therapies offer new treatment strategies in cartilage repair. For effective cartilage regeneration, the injectable hydrogel system needs to be bonded to the surrounding tissue and at the same time needs to be sufficiently stable for prolonged chondrogenesis. In this work, we utilised injectable hBMSC-laden microgels as the building blocks to create an assembled construct via N-hydroxysuccinimide-amine coupling. This crosslinking process also allows for rapid bonding between the assembled microgels and a surrounding tissue mimic. The resultant assembled microgel-construct provides both a physically stable and biologically dynamic environment for hBMSC chondrogenesis, leading to the production of a mature hyaline type cartilage structure.

Kartogenin Enhanced Chondrogenesis in Cocultures of Chondrocytes and Bone Mesenchymal Stem Cells.

Articular cartilage has poor capability of regeneration due to the avascular surrounding and low metabolic activity. Kartogenin (KGN), an emerging nonprotein heterocyclic compound, was screened to stimulate chondrogenic differentiation of bone mesenchymal stem cells (BMSCs). Coculturing BMSCs and chondrocytes was reported to overcome the shortcomings of forming fibroblastic and hypertrophic cartilages. In this study, KGN was incorporated into the Col-Tgel hydrogel to form a Gel/Cell/KGN complex, which fabricated an appropriate microenvironment for effective cartilage regeneration of BMSCs and/or chondrocytes. The complexes that incorporated KGN, BMSCs, and chondrocytes achieved higher lubricin expression and extracellular matrix production, such as characteristic glycosaminoglycans (GAGs) and collagen type II (COL II), compared to the monocultures of BMSCs or chondrocytes in vitro. The complexes compounding KGN, BMSCs, and chondrocytes (at an optimal ratio in the in vitro experiment) were transplanted into rat models to evaluate the repair effects. Our results suggested that the interaction between BMSCs and chondrocytes can substitute the use of growth factors to some degree and indicated the role of KGN in chondrogenesis induction. Besides, it is the first time (to our knowledge) that the expression of lubricin was found to be delayed in the coculture of mixed cells comparing with GAGs and COL II, which could be significant in cartilage tissue engineering.

Kartogenin-Incorporated Thermogel Supports Stem Cells for Significant Cartilage Regeneration.

Recently, cartilage tissue engineering (CTE) attracts increasing attention in cartilage defect repair. In this work, kartogenin (KGN), an emerging chondroinductive nonprotein small molecule, was incorporated into a thermogel of poly(L-lactide-co-glycolide)-poly(ethylene glycol)-poly(L-lactide-co-glycolide) (PLGA-PEG-PLGA) to fabricate an appropriate microenvironment of bone marrow mesenchymal stem cells (BMSCs) for effective cartilage regeneration. More integrative and smoother repaired articular surface, more abundant characteristic glycosaminoglycans (GAGs) and collagen II (COL II), and less degeneration of normal cartilage were obtained in the KGN and BMSCs coloaded thermogel group in vivo. In conclusion, the KGN-loaded PLGA-PEG-PLGA thermogel can be utilized as an alternative support for BMSCs to regenerate damaged cartilage in vivo.

Reversibly Immortalized Mouse Articular Chondrocytes Acquire Long-Term Proliferative Capability While Retaining Chondrogenic Phenotype.

Cartilage tissue engineering holds great promise for treating cartilaginous pathologies including degenerative disorders and traumatic injuries. Effective cartilage regeneration requires an optimal combination of biomaterial scaffolds, chondrogenic seed cells, and biofactors. Obtaining sufficient chondrocytes remains a major challenge due to the limited proliferative capability of primary chondrocytes. Here we investigate if reversibly immortalized mouse articular chondrocytes (iMACs) acquire long-term proliferative capability while retaining the chondrogenic phenotype. Primary mouse articular chondrocytes (MACs) can be efficiently immortalized with a retroviral vector-expressing SV40 large T antigen flanked with Cre/loxP sites. iMACs exhibit long-term proliferation in culture, although the immortalization phenotype can be reversed by Cre recombinase. iMACs express the chondrocyte markers Col2a1 and aggrecan and produce chondroid matrix in micromass culture. iMACs form subcutaneous cartilaginous masses in athymic mice. Histologic analysis and chondroid matrix staining demonstrate that iMACs can survive, proliferate, and produce chondroid matrix. The chondrogenic growth factor BMP2 promotes iMACs to produce more mature chondroid matrix resembling mature articular cartilage. Taken together, our results demonstrate that iMACs acquire long-term proliferative capability without losing the intrinsic chondrogenic features of MACs. Thus, iMACs provide a valuable cellular platform to optimize biomaterial scaffolds for cartilage regeneration, to identify biofactors that promote the proliferation and differentiation of chondrogenic progenitors, and to elucidate the molecular mechanisms underlying chondrogenesis.

Cartilage regeneration using biodegradable oxidized alginate/hyaluronate hydrogels

Despite the widespread use of alginate (AL) hydrogels in many biomedical applications, including tissue engineering, AL is inherently non-degradable under physiological conditions. We hypothesized that degradable alginate (dAL) would be useful for cartilage regeneration when combined with hyaluronate (HA). We prepared dAL by partial oxidation of AL using sodium periodate, and the degradation rate of AL hydrogel was able to be regulated by control of sodium periodate concentration. The degradable oxidized AL/HA gels were formed in the presence of cells and their characteristics were investigated. dAL/HA gels with primary chondrocytes were injected subcutaneously into mice. Effective cartilage regeneration was observed after 6 weeks of transplantation based on histological analysis. Moreover, substantial secretion of sulfated glycosaminoglycans and expression of chondrogenic marker genes were also observed compared with non-dAL/HA gels. These results indicate that dAL/HA hydrogels may be useful in cartilage regeneration, and in many tissue engineering applications.