Cartilage-like Tissue Research Articles

A Sendai virus-based expression system directs efficient induction of chondrocytes by transcription factor-mediated reprogramming.

Cartilage rarely heals spontaneously once damaged. Osteoarthritis (OA) is the most common degenerative joint disease among the elderly; however, effective treatment for OA is currently lacking. Autologous chondrocyte implantation (ACI), an innovative regenerative technology involving the implantation of healthy chondrocytes, may restore damaged lesions. Chondrocytes for ACI may potentially be induced from differentiated somatic cells using retrovirus (RV)-mediated transduction of three reprogramming factors (SOX9, KLF4, and c-MYC). However, the efficiency of the current induction system needs to be improved and the safety issues arising from the genomic integration of the vector DNA have to be addressed. To solve these problems, we used an RNA vector, termed the replication-defective and persistent Sendai virus vector (SeVdp), to express reprogramming factors for chondrocyte induction. Our results showed that the SeVdp-based vector induced chondrocytes more efficiently than the RV vector, probably because of robust and rapid expression of the transgenes, without any apparent integration of the SeVdp vector. The induced chondrocytes formed cartilage-like tissues when injected subcutaneously into mice. Thus, the SeVdp-based system for inducing chondrocytes may act as a foundation for developing safer and more effective treatments for damaged cartilage.

Second-look arthroscopy after ankle distraction arthroplasty

Background: Ankle distraction arthroplasty (ADA) is a method of treating terminal osteoarthritis of the ankle joint, which allows delaying fusion or total replacement. Aims: The aim of the study was to research the macroscopic image of the articular cartilage before and after ADA using second-look arthroscopy. Methods: A total of 17 ADA surgical interventions were performed. The average age was 48.5±13.57 years, among them 7 females (38.89%) and 10 males (61.11%). 4 patients underwent second-look arthroscopy of the ankle joint after ADA at different times. Results: All patients showed a statistically significant change in the functional outcome by 12 months from the moment of surgery, on the FAAM scale (p-value = 0.00060) and the AO FAS Ankle-hindfoot scale (as well as after removing the Ilizarov frame after 1, 3, 6, and 12 months). The reduction in pain was from 6.17 1.32 cm before surgery to 2 (1.4, 2.1) cm after 12 months. Conclusions: There were signs of regeneration of cartilaginous defects with their covering with cartilage-like soft tissue, which was probably responsible for the analgesic effect of ADA.

Rat Tracheal Cartilage Regeneration Using Mesenchymal Stem Cells Derived From Human iPS Cells.

Tracheal cartilage provides structural support to the airways to enable breathing. However, it can become damaged or impaired, sometimes requiring surgical resection and reconstruction. Previously, we clinically applied an artificial trachea composed of a polypropylene mesh and collagen sponge, with a favorable postoperative course. However, the artificial trachea presents a limitation, as the mesh is not biodegradable and cannot be used in pediatric patients. Compared to a polypropylene mesh, regenerated cartilage represents an ideal material for reconstruction of the damaged trachea. The use of mesenchymal stem cells (MSCs) as a source for cartilage regeneration has gained widespread acceptance, but challenges such as the invasiveness of harvesting and limited cell supply persist. Therefore, we focused on the potential of human-induced pluripotent stem cell (hiPSC)-derived mesenchymal stem cells (iMSCs) for tracheal cartilage regeneration. In this study, we aimed to regenerate tracheal cartilage on an artificial trachea as a preliminary step to replace the polypropylene mesh. iMSCs were induced from hiPSCs through neural crest cells and transplanted with a polypropylene mesh covered with a collagen sponge into the damaged tracheal cartilage in immunodeficient rats. Human nuclear antigen (HNA)-positive cells were observed in all six rats at 4 weeks and in six out of seven rats at 12 weeks after transplantation, indicating that transplanted iMSCs survived within the tracheal cartilage defects of rats. The HNA-positive cells coexpressed SOX9, and type II collagen was detected around HNA-positive cells in four of six rats at 4 weeks and in three of seven rats at 12 weeks after transplantation, reflecting cartilage-like tissue regeneration. These results indicate that the transplanted iMSCs could differentiate into chondrogenic cells and promote tracheal cartilage regeneration. iMSC transplantation thus represents a promising approach for human tracheal reconstruction.

Induction and real-time ultrasonic monitoring of 3D cartilage-like tissue by a low shear stresses-based bioreactor

Osteoarthritis is a significant socioeconomic illness that mainly affects the articular cartilage, a tissue with a low capacity for self-healing, making it an ideal target for regenerative medicine and tissue engineering. Current interventions to treat cartilage injuries may not be completely effective. In this study, we have developed a novel bioreactor that creates viscous shear stress by flow perfusion. This bioreactor could induce ex vivo maturation of biomimetic 3D cartilage scaffolds, providing a potential solution to this problem. Infrapatellar fat pad mesenchymal stem cells (IPFP-MSCs) were used as a cellular source of the functionalized 3D scaffolds made of 1,4-butanediol thermoplastic polyurethane (bTPUe) modified with pyrene butyric acid (PBA). Our results indicate that our bioreactor induced chondrogenic differentiation, as confirmed by DNA quantification, extracellular matrix determination, and metabolic assay, without any conditioned medium. To control the biomechanical stimulation on IPFP-MSCs, a low-intensity ultrasonic transmission system has been developed and embedded in the bioreactor. Combined with a finite element model (FEM), tissue growth and differentiation can be deconvoluted in real-time from the recorded ultrasonic propagation and interaction across the graft. The FEM reconstructs this complex interaction. This is the first time a low-shear stress-based bioreactor has been reported to not only induce chondrogenic evolution but also monitor it in real time.

In vitro and in vivo evaluation of periosteum-derived cells and iPSC-derived chondrocytes encapsulated in GelMA for osteochondral tissue engineering.

Osteochondral defects are deep joint surface lesions that affect the articular cartilage and the underlying subchondral bone. In the current study, a tissue engineering approach encompassing individual cells encapsulated in a biocompatible hydrogel is explored in vitro and in vivo. Cell-laden hydrogels containing either human periosteum-derived progenitor cells (PDCs) or human induced pluripotent stem cell (iPSC)-derived chondrocytes encapsulated in gelatin methacryloyl (GelMA) were evaluated for their potential to regenerate the subchondral mineralized bone and the articular cartilage on the joint surface, respectively. PDCs are easily isolated and expanded progenitor cells that are capable of generating mineralized cartilage and bone tissue in vivo via endochondral ossification. iPSC-derived chondrocytes are an unlimited source of stable and highly metabolically active chondrocytes. Cell-laden hydrogel constructs were cultured for up to 28 days in a serum-free chemically defined chondrogenic medium. On day 1 and day 21 of the differentiation period, the cell-laden constructs were implanted subcutaneously in nude mice to evaluate ectopic tissue formation 4 weeks post-implantation. Taken together, the data suggest that iPSC-derived chondrocytes encapsulated in GelMA can generate hyaline cartilage-like tissue constructs with different levels of maturity, while using periosteum-derived cells in the same construct type generates mineralized tissue and cortical bone in vivo. Therefore, the aforementioned cell-laden hydrogels can be an important part of a multi-component strategy for the manufacturing of an osteochondral implant.

Regeneration of amputated mice digit tips by including Wnt signaling pathway with CHIR99021 and IWP-2 chemicals in limb organ culture system.

Mammals have limited limb regeneration compared to amphibians. The role of Wnt signaling pathways in limb regeneration has rarely been studied. So, this study aimed to investigate the effect of Wnt-signaling using chemicals CHIR99021and IWP-2on amputated mice digit tips regeneration in aninvitroorgan culture system. The distal phalanx of paws from C57BL/6J mouse fetuses at E14.5, E16.5, and E18.5 was amputated.Then, the hands were cultured for 7 days.Subsequently, paws were treated with 1-50 µg/ml concentration of CHIR99021 and 5-10 µg/ml concentration of IWP-2. Finally, the new tissue regrowth was assessed by histological analysis, immunohistochemistry for BC, TCF1, CAN, K14, and P63 genes, and beta-catenin and Tcf1 genes were evaluated with RT-qPCR. The paws of E14.5 and E16.5 days were shrinkaged and compressed after 7 days, so the paws of 18.5E that were alive were selected.As a result, newly-grown masses at digit tips were observed in 25 and 30 µl/ml concentrations of the CHR99021 group but not in the IWP2 treatment (*P<0.05; **P<0.01). qRT-PCR analysis confirmed the significant up-regulation of beta-catenin and Tcf1 genes in CHIR99021 group in comparison to the IWP-2 group (P<0.05). Moreover, Alcian-blue staining demonstrated the presence of cartilage-like tissue at regenerated mass in the CHIR group. In immunohistochemistry analysis beta-catenin, ACN, Keratin-14, and P63 protein expression were observed in digit tips in the CHIR-treated group. By activating the Wnt signaling pathway, cartilage-like tissue formed in the blastema-like mass in the mouse's amputated digit tips.

Spider silk enhanced tissue engineering of cartilage tissue: Approach of a novel bioreactor model using adipose derived stromal cells.

Human cartilage tissue remains a challenge for the development of therapeutic options due to its poor vascularization and reduced regenerative capacities. There are a variety of research approaches dealing with cartilage tissue engineering. In addition to different biomaterials, numerous cell populations have been investigated in bioreactor-supported experimental setups to improve cartilage tissue engineering. The concept of the present study was to investigate spider silk cocoons as scaffold seeded with adipose-derived stromal cells (ASC) in a custom-made bioreactor model using cyclic axial compression to engineer cartilage-like tissue. For chemical induction of differentiation, BMP-7 and TGF-β2 were added and changes in cell morphology and de-novo tissue formation were investigated using histological staining to verify chondrogenic differentiation. By seeding spider silk cocoons with ASC, a high colonization density and cell proliferation could be achieved. Mechanical induction of differentiation using a newly established bioreactor model led to a more roundish cell phenotype and new extracellular matrix formation, indicating a chondrogenic differentiation. The addition of BMP-7 and TGF-β2 enhanced the expression of cartilage specific markers in immunohistochemical staining. Overall, the present study can be seen as pilot study and valuable complementation to the published literature.

Structural and Molecular Changes of Human Chondrocytes Exposed to the Rotating Wall Vessel Bioreactor.

Over the last 30 years, the prevalence of osteoarthritis (OA), a disease characterized by a loss of articular cartilage, has more than doubled worldwide. Patients suffer from pain and progressive loss of joint function. Cartilage is an avascular tissue mostly consisting of extracellular matrix with embedded chondrocytes. As such, it does not regenerate naturally, which makes an early onset of OA prevention and treatment a necessity to sustain the patients' quality of life. In recent years, tissue engineering strategies for the regeneration of cartilage lesions have gained more and more momentum. In this study, we aimed to investigate the scaffold-free 3D cartilage tissue formation under simulated microgravity in the NASA-developed rotating wall vessel (RWV) bioreactor. For this purpose, we cultured both primary human chondrocytes as well as cells from the immortalized line C28/I2 for up to 14 days on the RWV and analyzed tissue morphology, development of apoptosis, and expression of cartilage-specific proteins and genes by histological staining, TUNEL-assays, immunohistochemical detection of collagen species, and quantitative real-time PCR, respectively. We observed spheroid formation in both cell types starting on day 3. After 14 days, constructs from C28/I2 cells had diameters of up to 5 mm, while primary chondrocyte spheroids were slightly smaller with 3 mm. Further inspection of the 14-day-old C28/I2 spheroids revealed a characteristic cartilage morphology with collagen-type 1, -type 2, and -type 10 positivity. Interestingly, these tissues were less susceptible to RWV-induced differential gene expression than those formed from primary chondrocytes, which showed significant changes in the regulation of IL6, ACTB, TUBB, VIM, COL1A1, COL10A1, MMP1, MMP3, MMP13, ITGB1, LAMA1, RUNX3, SOX9, and CASP3 gene expression. These diverging findings might reflect the differences between primary and immortalized cells. Taken together, this study shows that simulated microgravity using the RWV bioreactor is suitable to engineer dense 3D cartilage-like tissue without addition of scaffolds or any other artificial materials. Both primary articular cells and the stable chondrocyte cell line C28/I2 formed 3D neocartilage when exposed for 14 days to an RWV.

Scaffolded spheroids as building blocks for bottom-up cartilage tissue engineering show enhanced bioassembly dynamics

Due to the capability of cell spheroids (SPH) to assemble into large high cell density constructs, their use as building blocks attracted a lot of attention in the field of biofabrication. Nevertheless, upon maturation, the composition along with the size of such building blocks change, affecting their fusiogenic ability to form a cohesive tissue construct of controllable size. This natural phenomenon remains a limitation for the standardization of spheroid-based therapies in the clinical setting.We recently showed that scaffolded spheroids (S-SPH) can be produced by forming spheroids directly within porous PCL-based microscaffolds fabricated using multiphoton lithography (MPL). In this new study, we compare the bioassembly potential of conventional SPHs versus S-SPHs depending on their degree of maturation. Doublets of both types of building blocks were cultured and their fusiogenicity was compared by measuring the intersphere angle, the length of the fusing spheroid pairs (referred to as doublet length) as well as their spreading behaviour. Finally, the possibility to fabricate macro-sized tissue constructs (i.e. cartilage-like) from both chondrogenic S-SPHs and SPHs was analyzed.This study revealed that, in contrast to conventional SPHs, S-SPHs exhibit robust and stable fusiogenicity, independently from their degree of maturation. In order to understand this behavior, we further analyze the intersection area of doublets, looking at the kinetic of cell migration and at the mechanical stability of the formed tissue using dissection measurements. Our findings indicate that the presence of microscaffolds enhances the ability of spheroids to be used as building blocks for bottom-up tissue engineering, which is an important advantage compared to conventional spheroid-based therapy approaches. Statement of significanceThe approach of using SPHs as building blocks for bottom-up tissue engineering offers a variety of advantages. At the same time the self-assembly of large tissues remains challenging due to several intrinsic properties of SPHs, such as for instance the shrinkage of tissues assembled from SPHs, or the reduced fusiogenicity commonly observed with mature SPHs. In this work, we demonstrate the capability of scaffolded spheroids (S-SPH) to fuse and recreate cartilage-like tissue constructs despite their advanced maturation stage. In this regard, the presence of microscaffolds compensates for some of the intrinsic limitations of SPHs and can help to overcome current limitations of spheroid-based tissue engineering.

Atelocollagen Scaffold Enhances Cartilage Regeneration in Osteochondral Defects: A Study in Rabbits.

To enhance articular cartilage healing, microfractures (Mfx) and bone marrow aspirate concentrate (BMAC) are commonly used, and some form of scaffold is often used together to increase its efficacy. Herein, we compared the efficacy of atelocollagen scaffold to that of collagen scaffold when used with Mfx or BMAC on osteochondral defect of animal. This experiment was designed in two stages, and therapeutic effects of Mfx and BMAC were respectively evaluated when used with atelocollagen or collagen scaffold. Femoral condyle defects were artificially created in male New Zealand White rabbits, and in each stage, 12 rabbits were randomly allocated into three treatment groups: test group with additional atelocollagen scaffold, the positive control group with collagen scaffold, and the negative control group. Then, for 12weeks, macroscopic and histological evaluations were performed. At 12weeks, defects in the test group were fully regenerated with normal cartilage-like tissue, and were well integrated with the surrounding cartilage at both stages experiment, whereas defects in the control groups were not fully filled with regenerated tissue, and the tissue appeared as fibrous tissue. Histologically, the regenerated tissue in the test group showed a statistically significant improvement compared to the positive and negative control groups, achieving a similar structure as normal articular cartilage. The results showed that implantation of the atelocollagen scaffold enhanced cartilage regeneration following osteochondral defects in rabbits. This suggests that the atelocollagen scaffold can be used with Mfx or BMAC for effective regeneration of osteochondral defects.

Directed differentiation of human pluripotent stem cells into articular cartilage reveals effects caused by absence of WISP3, the gene responsible for progressive pseudorheumatoid arthropathy of childhood

ObjectivesProgressive pseudorheumatoid arthropathy of childhood (PPAC), caused by deficiency of WNT1 inducible signalling pathway protein 3 (WISP3), has been challenging to study because no animal model of the disease exists...

Selective Extracellular Matrix Guided Mesenchymal Stem Cell Self-Aggregate Engineering for Replication of Meniscal Zonal Tissue Gradient in a Porcine Meniscectomy Model.

Degenerative meniscus tears (DMTs) are prevalent findings in osteoarthritic knees, yet current treatment is mostly limited to arthroscopic partial meniscectomy rather than regeneration, which further exacerbates arthritic changes. Translational research regarding meniscus regeneration is hindered by the complex, composite nature of the meniscus which exhibit a gradient from inner cartilage-like tissue to outer fibrous tissue, as well as engineering hurdles often requiring growth factors and cross-linking agents. Here, a meniscus zonal tissue gradient is proposed using zone-specific decellularized meniscus extracellular matrix (DMECM) and autologous synovial mesenchymal stem cells (SMSC) via self-aggregation without the use of growth factors or cross-linking agents. Combination with zone-specific DMECM during self-aggregation of MSCs forms zone-specific meniscus tissue that reflects the respective DMECM harvest site. The implantation of these constructs leads to the regeneration of meniscus tissue resembling the native meniscus, demonstrating inner cartilaginous and outer fibrous characteristics as well as recovery of native meniscal microarchitecture in a porcine partial meniscectomy model at 6 months. In all, the findings offer a potential regenerative therapy for DMTs that may improve current partial meniscectomy-based patient care.

Synovial chondromatosis of the temporomandibular joint

When cartilage-like tissue is present in the synovial membrane of joints, it is known as synovial chondromatosis (SC). The main cause is believed to be the metaplasia of embryonal mesenchymal tissue in the synovial membrane. The metaplastic foci grow within the joint space and can detach from the synovial wall, forming free-floating bodies. While SC is more commonly associated with trauma in the temporomandibular joint (TMJ), the exact relationship between trauma and SC remains unclear. Improved diagnostic techniques, such as CT and MRI, have led to increased recognition of SC in the TMJ. Gender differences in the prevalence of TMJ SC have been observed, with women being more affected. Diagnosis involves imaging techniques like X-rays, CT scans, MRIs, and surgical removal of loose bodies is the preferred treatment. Arthroscopy is recommended for small, isolated loose bodies, while open surgery may be necessary for complete removal. Long-term follow-up is essential to monitor for recurrence. Histopathological findings reveal a mixture of cartilage and bone, with ossification and calcification present.

Potential function of Scx+/Sox9+ cells as progenitor cells in rotator cuff tear repair in rats

Tendons and their attachment sites to bone, fibrocartilaginous tissues, have poor self-repair capacity when they rupture, and have risks of retear even after surgical repair. Thus, defining mechanisms underlying their repair is required in order to stimulate tendon repairing capacity. Here we used a rat surgical rotator cuff tear repair model and identified cells expressing the transcription factors Scleraxis (Scx) and SRY-box 9 (Sox9) as playing a crucial role in rotator cuff tendon-to-bone repair. Given the challenges of establishing stably reproducible models of surgical rotator cuff tear repair in mice, we newly established Scx-GFP transgenic rats in which Scx expression can be monitored by GFP. We observed tissue-specific GFP expression along tendons in developing ScxGFP transgenic rats and were able to successfully monitor tissue-specific Scx expression based on GFP signals. Among 3-, 6-, and 12-week-old ScxGFP rats, Scx+/Sox9+ cells were most abundant in 3-week-old rats near the site of humerus bone attachment to the rotator cuff tendon, while we observed significantly fewer cells in the same area in 6- or 12-week-old rats. We then applied a rotator cuff repair model using ScxGFP rats and observed the largest number of Scx+/Sox9+ cells at postoperative repair sites of 3-week-old relative to 6- or 12-week-old rats. Tendons attach to bone via fibrocartilaginous tissue, and cartilage-like tissue was seen at repair sites of 3-week-old but not 6- or 12-week-old rats during postoperative evaluation. Our findings suggest that Scx+/Sox9+ cells may function in rotator cuff repair, and that ScxGFP rats could serve as useful tools to develop therapies to promote rotator cuff repair by enabling analysis of these activities.

Humeral Tissue Remodeling in Contact with Pyrocarbon Interposition Implant

The interactions between pyrocarbon (PyC) interposition implants and human humeral cancellous bone leads to the formation of a new interpositional tissue made a dense layer of cortical bone and a cartilage-like tissue. A sample of the tissue in contact with the PyC interposition implant was harvested on the humeral bone side from 6 patients undergoing a re-operation for implant removal due to staged surgery or clinical failure related to pain. The samples where then prepared for histology to evaluate the tissue morphology and immunohistology to assess for the presence of collagen I and collagen II within the matrix. For all the cases, a layer of dense cortical bone and a layer of soft tissue was observed between the PyC interpositional implant and the humeral cancellous bone. The nature of the interpositional soft tissue was different between the cases. While the hypothesis that a new tissue was modeled at PyC - humeral cancellous bone interface was confirmed, the nature of the soft interpositional tissue evolved from a fibrocellular to a cartilage-like tissue depending on the cases. The hypotheses for the different nature of the soft interpositional tissue observed between the different cases was based on humeral bone mechanobiological remodeling. The influence of the degree of attachment between the soft and bony interpositional-tissues and on the PISA geometry on the soft interpositional tissue nature are discussed. Further investigations on the role of humeral bone and on the PISA biomechanics should be performed to better understand this tissue remodeling.

Bioactivity of human adult stem cells and functional relevance of stem cell-derived extracellular matrix in chondrogenesis

BackgroundAutologous chondrocyte implantation (ACI) has been used to treat articular cartilage defects for over two decades. Adult stem cells have been proposed as a solution to inadequate donor cell numbers often encountered in ACI. Multipotent stem/progenitor cells isolated from adipose, bone marrow, and cartilage are the most promising cell therapy candidates. However, different essential growth factors are required to induce these tissue-specific stem cells to initiate chondrogenic differentiation and subsequent deposition of extracellular matrix (ECM) to form cartilage-like tissue. Upon transplantation into cartilage defects in vivo, the levels of growth factors in the host tissue are likely to be inadequate to support chondrogenesis of these cells in situ. The contribution of stem/progenitor cells to cartilage repair and the quality of ECM produced by the implanted cells required for cartilage repair remain largely unknown. Here, we evaluated the bioactivity and chondrogenic induction ability of the ECM produced by different adult stem cells.MethodsAdult stem/progenitor cells were isolated from human adipose (hADSCs), bone marrow (hBMSCs), and articular cartilage (hCDPCs) and cultured for 14 days in monolayer in mesenchymal stromal cell (MSC)–ECM induction medium to allow matrix deposition and cell sheet formation. The cell sheets were then decellularized, and the protein composition of the decellularized ECM (dECM) was analyzed by BCA assay, SDS-PAGE, and immunoblotting for fibronectin (FN), collagen types I (COL1) and III (COL3). The chondrogenic induction ability of the dECM was examined by seeding undifferentiated hBMSCs onto the respective freeze-dried solid dECM followed by culturing in serum-free medium for 7 days. The expression levels of chondrogenic genes SOX9, COL2, AGN, and CD44 were analyzed by q-PCR.ResultshADSCs, hBMSCs, and hCDPCs generated different ECM protein profiles and exhibited significantly different chondrogenic effects. hADSCs produced 20–60% more proteins than hBMSCs and hCDPCs and showed a fibrillar-like ECM pattern (FNhigh, COL1high). hCDPCs produced more COL3 and deposited less FN and COL1 than the other cell types. The dECM derived from hBMSCs and hCDPCs induced spontaneous chondrogenic gene expression in hBMSCs.ConclusionsThese findings provide new insights on application of adult stem cells and stem cell-derived ECM to enhance cartilage regeneration.

Long-Term Culture Performance of a Polyelectrolyte Complex Microcapsule Platform for Hyaline Cartilage Repair.

Articular cartilage (AC) tissue repair and regeneration remains an ongoing challenge. One component of the challenge is the limited ability to scale an engineered cartilage graft to clinically relevant sizes while maintaining uniform properties. In this paper, we report on the evaluation of our polyelectrolyte complex microcapsule (PECM) platform technology as a technique for generating cartilage-like spherical modules. Bone marrow-derived mesenchymal stem cells (bMSCs) or primary articular chondrocytes were encapsulated within PECMs composed of methacrylated hyaluronan, collagen I, and chitosan. The formation of cartilage-like tissue in the PECMs over a 90-day culture was characterized. The results showed that chondrocytes exhibited superior growth and matrix deposition compared to either chondrogenically-induced bMSCs or a mixed PECM culture containing both chondrocytes and bMSCs. The chondrocyte-generated matrix filled the PECM and produced substantial increases in capsule compressive strength. The PECM system thus appears to support intracapsular cartilage tissue formation and the capsule approach promotes efficient culture and handling of these micro tissues. Since previous studies have proven the feasibility of fusing such capsules into large tissue constructs, the results suggest that encapsulating primary chondrocytes in PECM modules may be a viable route toward achieving a functional articular cartilage graft.

Bioinspired collagen-gelatin-hyaluronic acid-chondroitin sulfate tetra-copolymer scaffold biomimicking native cartilage extracellular matrix facilitates chondrogenesis of human synovium-derived stem cells

The microenvironment plays a crucial role in stem cell differentiation, and a scaffold that mimics native cartilaginous extracellular components can promote chondrogenesis. In this study, a collagen–gelatin–hyaluronic acid–chondroitin sulfate tetra-copolymer scaffold with composition and architecture similar to those of hyaline cartilage was fabricated using a microfluidic technique and compared with a pure gelatin scaffold. The newly designed biomimetic scaffold had a swelling ratio of 1278 % ± 270 %, a porosity of 77.68 % ± 11.70 %, a compressive strength of 1005 ± 174 KPa, and showed a good resilience against compression force. Synovium-derived stem cells (SDSCs) seeded into the tetra-copolymer scaffold attached to the scaffold firmly and exhibited good mitochondrial activity, high cell survival with a pronounced glycosaminoglycan production. SDSCs cultured on the tetra-copolymer scaffold with chondrogenic induction exhibited upregulated mRNA expression of COL2A1, ChM-1, Nrf2, TGF-β1, and BMP-7. Ex vivo study revealed that the SDSC–tetra-copolymer scaffold regenerated cartilage-like tissue in SCID mice with abundant type II collagen and S-100 production. BMP7 and COL2A1 expression in the tetra-copolymer scaffold group was much higher than that in the gelatin scaffold group ex vivo. The tetra-copolymer scaffold thus exhibits strong chondrogenic capability and will facilitate cartilage tissue engineering.

HIGH-RESOLUTION 3D-PRINTED MICROSCAFFOLDS FOR BOTTOM-UP CARTILAGE TISSUE ENGINEERING

In the field of tissue engineering (TE), mainly two approaches have been widely studied and utilised throughout the last two decades. Ovsianikov et al. proposed a third strategy for tissue engineering to combine the advantages of the scaffold-based and scaffold-free approach [1].We utilise the third strategy for TE by fabrication of cell spheroids that are reinforced by microscaffolds, called tissue units (TUs). Aim of the presented study is to differentiate TUs towards a chondrogenic phenotype to show the self-assembly of a millimetre sized cartilage-like tissue in a bottom-up TE approach in vitro.Two-Photon polymerization (2PP) was utilised to fabricate highly porous microscaffolds with a diameter of 300 µm. The biocompatible and biodegradable, resin Degrad INX (supplied from Xpect INX, Ghent, Belgium) was used for 3D-printing. Each microscaffold was seeded with 4000 human adipose derived stem cells (hASCs) in low-adhesive 96-well plates to allow spheroid formation. TUs were differentiated towards the chondrogenic lineage by application of chondrogenic media, subsequently merged in a cylindrical agarose mold, to fuse into a connected tissue with a diameter of ~1.8 mm and a height of 8 mm.The characterization of TUs differentiated towards the chondrogenic phenotype included gene expression and protein analysis. Furthermore, immunohistochemically staining for Collagen II and Alcian blue staining were performed to investigate the matrix deposition and fusion of the self-assembled tissue.Our results suggest that the utilised method could be a promising approach for a variety of tissue engineering approaches, due to the good applicability to a defect side combined with the self-assembly properties of the TUs. Furthermore, the differentiation potential of hASCs is not limited to chondrogenic lineages only, which could pave the way to further TE applications in the future.Acknowledgements:This research work was financially supported by the European Research Council (Consolidator Grant 772464 A.O.)

MUSSEL ADHESIVE PROTEIN-BASED ADHESIVE TO RETAIN STEM CELLS FOR CARTILAGE REGENERATION

In this study, we developed biocompatible adhesive which enables implanted chondrogenic-enhanced hASCs being strongly fixed to the lesion site of defected cartilage.The bioengineered mussel adhesive protein (MAP) was produced and purified using a bacterial expression system as previously reported. The cell encapsulated coacervate was formulated with two polyelectrolyte, the MAP and 723kDa hyaluronic acid (HA). MAP formed liquid microdroplets with HA and subsequently gelated into microparticles, which is highly viscous and strongly adhesive.The MAP with chondro-induced hASCs were implanted on the osteochondral defect created in the patellar groove/condyle of OA-induced rabbits. Rabbits were allocated to three different groups as follows: Group1 – Fibrin only; Group2 – Fibrin with hASCs (1.5×106 chondro-induced hASCs); Group3; MAP with hASCs.The implanted cells were labeled with a fluorescent dye for in vivo visualization. After 35 days, fluorescent signals were more potently detected for MAP with hASCs group than Fibrin with hASCs group in osteochondral defect model. Moreover, histological assessment showed that MAP with hASCs group had the best healing and covered with hyaline cartilage-like tissue. The staining image shows that MAP with hASCs group were filled with perfectly differentiated chondrocytes. Although Fibrin with hASCs group had better healing than fibrin only group, it was filled with fibrous cartilage which owes its flexibility and toughness. As MAP with hASCs group has higher possibility of differentiating to complete cartilage, Fibrin only group and Fibrin with hASCs group have failed to treat OA by rehabilitating cartilage. In order to clarify the evidence of remaining human cell proving efficacy of newly developed bioadhesive, human nuclear staining was proceeded with sectioned rabbit cartilage tissue. The results explicitly showed MAP with hASCs group have retained more human cells than Fibrin only and Fibrin with hASCs groups.We investigated the waterproof bioadhesive supporting transplanted cells to attach to defect lengthily in harsh environment, which prevents cells from leaked to other region of cartilage. Collectively, the newly developed bio-adhesive, MAP, could be successfully applied in OA treatment as a waterproof bioadhesive with the capability of the strong adhesion to target defect sites.