Hyaline-like Cartilage Regeneration Research Articles

Intra-Articular Mesenchymal Stem Cell Injection for Knee Osteoarthritis: Mechanisms and Clinical Evidence

Knee osteoarthritis presents higher incidences than other joints, with increased prevalence during aging. It is a progressive process and may eventually lead to disability. Mesenchymal stem cells (MSCs) are expected to repair damaged issues due to trilineage potential, trophic effects, and immunomodulatory properties of MSCs. Intra-articular MSC injection was reported to treat knee osteoarthritis in many studies. This review focuses on several issues of intra-articular MSC injection for knee osteoarthritis, including doses of MSCs applied for injection and the possibility of cartilage regeneration following MSC injection. Intra-articular MSC injection induced hyaline-like cartilage regeneration, which could be seen by arthroscopy in several studies. Additionally, anatomical, biomechanical, and biochemical changes during aging and other causes participate in the development of knee osteoarthritis. Conversely, appropriate intervention based on these anatomical, biomechanical, biochemical, and functional properties and their interactions may postpone the progress of knee OA and facilitate cartilage repair induced by MSC injection. Hence, post-injection rehabilitation programs and related mechanisms are discussed.

A Photoannealed Granular Hydrogel Facilitating Hyaline Cartilage Regeneration via Improving Chondrogenic Phenotype.

Hydrogel-based chondrocyte implantation presents a promising tissue engineering strategy for cartilage repair. However, the widely used elastic hydrogels usually restrict cell volume expansion and induce the dedifferentiation of encapsulated chondrocytes. To address this limitation, a photoannealed granular hydrogel (GH) composed of hyaluronic acid, polyethylene glycol, and gelatin was formulated for cartilage regeneration in this study. The unannealed GH prepared by Diels-Alder cross-linked microgels could be mixed with chondrocytes and delivered to cartilage defects by injection, after which light was introduced to anneal the scaffold, leading to the formation of a stable and microporous chondrocyte deploying scaffold. The in vitro studies showed that GH could promote the volume expansion and morphology recovery of chondrocytes and significantly improve their chondrogenic phenotype compared to the nongranular hydrogel (nGH) with similar compositions. Further in vivo studies of subcutaneous culture and the rat full-thickness cartilage defect model proved that chondrocyte loaded GH could significantly stimulate hyaline cartilage matrix deposition and connection, therefore facilitating hyaline-like cartilage regeneration. Finally, the mechanistic study revealed that GH might improve chondrogenic phenotype via activating the AMP-activated protein kinase/glycolysis axis. This study proves the great feasibility of GHs as in situ chondrocyte deploying scaffolds for cartilage regeneration and brings new insights in designing hydrogel scaffold for cartilage tissue engineering.

Double – network hydrogel based on exopolysaccharides as a biomimetic extracellular matrix to augment articular cartilage regeneration

Cartilage regeneration remains a current challenge with no satisfactory strategy in surgery. Hydrogels with structurally and biochemically biomimicking characteristics have been regarded as a promising approach for the success of cartilage regeneration. Naturally sourced hydrogels from exopolysaccharides are ideal candidates for the construction of biomimetic extracellular matrix (ECM) because of their biomimetic networks, high water content, cytocompatibility, and biodegradability. Here, an approach that integrates covalent and ionic bonds in a hydrogel system is shown to form a natural polymeric hydrogel double network (DN) for promoting the adhesion and proliferation of chondrocytes and supporting the formation of matured cartilage tissue. DN hydrogels comprised of chemically crosslinked hyaluronan (HA) and physically crosslinked gellan gum (GG) were developed for potential scaffold fabrication. Compared with HA single network (SN) hydrogel and GG SN hydrogel, the obtained HA/GG DN hydrogel with Young's modulus of 28.6 kPa exhibited adequate compressive strength (208.9 kPa) and high toughness (dissipated energy 2837 J/m3) and thus can be used as a biomimetic extracellular matrix for minimal invasively repairing cartilage. In vitro studies showed that HA/GG DN hydrogel-based ECM promoted the proliferation of chondrocytes. The HA/GG DN hydrogel significantly supported the deposition of cartilage ECM-specific sulfated glycosaminoglycan and type II collagen and facilitated the formation of cartilage tissues. In a rabbit osteochondral defect model, HA/GG DN hydrogel significantly improved cartilage regeneration. The HA/GG DN hydrogel as a biomimetic ECM is a promising candidate as a biomaterial scaffold for cartilage regeneration and repair. Statement of significanceThe fabrication of a biomaterial scaffold as an artificial extracellular matrix (ECM) for cartilage regeneration remains a big challenge. In this work, we fabricated a double-network (DN) hydrogel based on hyaluronan and gellan gum (HA/GG) through a sequential chemical and physical cross-linking process. The HA/GG DN hydrogel exhibited high compressive strength, high toughness, stiffness, and good self-recovery property. The HA/GG DN hydrogel can support chondrocyte proliferation and new ECM deposition correlated with the enhanced mechanical properties, good cytocompatibility, and biodegradability. In vivo animal experiments demonstrated that this HA/GG DN hydrogel facilitates hyaline-like cartilage regeneration. These findings imply that the developed HA/GG DN hydrogel as a biomimetic ECM offers a hopeful new platform for cartilage tissue engineering.

Preparation of Assemblable Chondral and Subchondral Bone Microtissues for Osteochondral Tissue Engineering.

Microtissues exhibit great advantages in injecting with minimum invasiveness, mimicking natural tissues, and promoting tissue regeneration. However, very few studies have focused on the construction of osteochondral microtissues that could simultaneously support hyaline-like cartilage and bone tissue regeneration. In this study, chondral microtissues that could favor the formation of hyaline-like cartilages and subchondral bone microtissues that could repair subchondral defects to support the neo-generated cartilages were successfully constructed for osteochondral tissue engineering. For chondral repair, the developed chondral microgels with high porosity and hydrophilicity could make cells spherical, favor the formation of cell aggregates, and show an excellent differentiation effect toward hyaline-like cartilage, thus contributing to the production of chondral microtissues. For subchondral bone repair, the fabricated subchondral microgels realize cell adhesion and proliferation and support the osteogenic differentiation of stem cells, thus favoring the formation of subchondral bone microtissues. The injectable chondral and subchondral bone microtissues could be stably assembled by Michael addition reaction between sulfhydryl groups of microtissues and double bonds of hydrophilic macromolecular cross-linker. At 12 weeks postimplantation, osteochondral microtissues could support the reconstruction of osteochondral-like tissues. The present study provides new insight into the microtissues for repair of osteochondral tissues.

The Beneficial Effect of an Intra-articular Injection of Losartan on Microfracture-Mediated Cartilage Repair Is Dose Dependent

Background: A previous publication demonstrated that the oral intake of losartan promoted microfracture-mediated hyaline-like cartilage repair in osteochondral defects of a rabbit knee model. However, an intra-articular (IA) injection of losartan may have direct beneficial effects on cartilage repair and has not been studied. Purpose: To determine the dosage and beneficial effects of an IA injection of losartan on microfracture-mediated cartilage repair and normal cartilage homeostasis. Study Design: Controlled laboratory study. Methods: Rabbits were divided into 5 groups (n = 6 each): a microfracture group (MFX group) and 4 different losartan treatment groups that received varying doses of IA losartan (0.1, 1, 10, and 100 mg per knee). An osteochondral defect (5 mm) was created in the trochlear groove cartilage of 1 limb in each rabbit, and 5 microfracture perforations were made in the osteochondral defect. Both the injured and the contralateral knee joints were injected with IA losartan immediately after microfracture and at 2 and 4 weeks after surgery. Rabbits were sacrificed at 6 weeks after surgery for analysis including gross observation, micro–computed tomography, histology, and reverse transcription quantitative polymerase chain reaction. Results: Micro–computed tomography and gross observation demonstrated comparable subchondral bone healing and hyaline-like cartilage morphology in the 0.1-, 1-, and 10-mg losartan groups relative to the MFX group. Conversely, the 100-mg losartan group showed neither bony defect healing nor cartilage repair. Histology revealed higher O’Driscoll scores and hyaline-like cartilage regeneration in the 1-mg losartan group compared with the MFX group. In contrast, the 100-mg losartan group showed the lowest histology score and no cartilage repair. An IA injection of losartan at the doses of 0.1, 1, and 10 mg did not cause adverse effects on uninjured cartilage, while the 100-mg dose induced cartilage damage. Quantitative polymerase chain reaction results showed downregulation of the transforming growth factor β (TGF-β) signaling pathway after IA losartan injection. Conclusion: An IA injection of losartan at the dose of 1 mg was most effective for the enhancement of microfracture-mediated cartilage repair without adversely affecting uninjured cartilage. Conversely, a high dose (100 mg) IA injection of losartan inhibited cartilage repair in the osteochondral defect and was chondrotoxic to normal articular cartilage. Clinical Relevance: An IA injection of losartan at an optimal dosage represents a novel microfracture enhancement therapy and warrants a clinical trial for future clinical applications.

Ascorbic Acid and Iron Supplement Treatment Improves Stem Cell–Mediated Cartilage Regeneration in a Minipig Model

Background: The transplantation of mesenchymal stem cells (MSCs) into cartilage defects has led to variable cartilage repair outcomes. Previous in vitro studies have shown that ascorbic acid and reduced iron independently can improve the chondrogenic differentiation of MSCs. However, the combined effect of ascorbic acid and iron supplementation on MSC differentiation has not been investigated. Purpose: To investigate the combined in vivo effects of ascorbic acid and a US Food and Drug Administration (FDA)–approved iron supplement on MSC-mediated cartilage repair in mature Göttingen minipigs. Study Design: Controlled laboratory study. Methods: We pretreated bone marrow–derived MSCs with ascorbic acid and the FDA-approved iron supplement ferumoxytol and then transplanted the MSCs into full-thickness cartilage defects in the distal femurs of Göttingen minipigs. Untreated cartilage defects served as negative controls. We evaluated the cartilage repair site with magnetic resonance imaging at 4 and 12 weeks after MSC implantation, followed by histological examination and immunofluorescence staining at 12 weeks. Results: Ascorbic acid plus iron–pretreated MSCs demonstrated a significantly better MOCART (magnetic resonance observation of cartilage repair tissue) score (73.8 ± 15.5), better macroscopic cartilage regeneration score according to the International Cartilage Repair Society (8.6 ± 2.0), better Pineda score (2.9 ± 0.8), and larger amount of collagen type II (28,469 ± 21,313) compared with untreated controls (41.3 ± 2.5, 1.8 ± 2.9, 12.8 ± 1.9, and 905 ± 1326, respectively). The obtained scores were also better than scores previously reported in the same animal model for MSC implants without ascorbic acid. Conclusion: Pretreatment of MSCs with ascorbic acid and an FDA-approved iron supplement improved the chondrogenesis of MSCs and led to hyaline-like cartilage regeneration in the knee joints of minipigs. Clinical Relevance: Ascorbic acid and iron supplements are immediately clinically applicable. Thus, these results, in principle, could be translated into clinical applications.

Exploiting Joint-Resident Stem Cells by Exogenous SOX9 for Cartilage Regeneration for Therapy of Osteoarthritis.

The lack of effective treatment options for osteoarthritis (OA) is mostly due to the very limited regenerative capacity of articular cartilage. Mesenchymal stem cells (MSCs) have been most extensively explored for cell-based therapy to induce cartilage regeneration for OA. However, current in vitro expanded MSC-based approaches have significant drawbacks. On the other hand, osteoarthritic joints contain chondrocyte progenitors and MSCs in several niches which have the potential yet fail to differentiate into chondrocytes for cartilage regeneration. One of the underlying mechanisms of the failure is that these chondrocyte progenitors and MSCs in OA joints are deficient in the activity of chondrogenic transcription factor SOX9 (SRY-type high-mobility group box-9). Thereby, replenishing with exogenous SOX9 would reactivate the potential of these stem cells to differentiate into chondrocytes. Cell-permeable, super-positively charged SOX9 (scSOX9) protein is able to promote hyaline-like cartilage regeneration by inducing chondrogenic differentiation of bone marrow derived MSCs in vivo. This scSOX9 protein can be administered into osteoarthritic joints by intra-articular injection. This one-step, cell-free supplement of exogenous SOX9 may harness the regenerative potential of the intrinsic MSCs within the joint cavity to stimulate cartilage regeneration in OA.

Long-term durable repaired cartilage induced by SOX9 in situ with bone marrow-derived mesenchymal stem cells.

Background: Microfracture is a common procedure for cartilage repair, but it often produces inferior fibrocartilage. We previously reported that a super positively charged SOX9 (scSOX9) promoted hyaline-like cartilage regeneration by inducing bone marrow derived mesenchymal stem cell differentiation into chondrocytes in vivo. Here we examined the long-term efficacy of cartilage repair induced by microfracture with scSOX9 by assessing the biomechanical property of the repaired cartilage.Methods: A cartilage defect was created at the right femoral trochlear groove in New Zealand female rabbits and microfracture was performed. The scSOX9 protein was administered at the site of microfracture incorporated in a collagen membrane.Results: At 12 and 24 weeks, scSOX9 treatment induced hyaline-like cartilage while collagen-membrane alone induced fibrocartilage and mutant scSOX9-A76E poorly induced cartilage repair. The cartilage matrix in scSOX9-treated group showed highly enriched proteoglycan content. Consistent with the histological feature and the thickness of the repaired cartilage, the mechanical property of scSOX9-induced cartilage was also similar to that of normal cartilage.Conclusion: This long-term in vivo study demonstrated that in combination with microfracture, scSOX9 was able to induce reparative tissue with features of hyaline cartilage which was durable in long-term. This technology has the potential to translate into clinical use for cartilage repair to prevent progression to osteoarthritis.

Arthroscopic Chondral Defect Repair With Extracellular Matrix Scaffold and Bone Marrow Aspirate Concentrate

Chondral defects of the knee are prevalent and often encountered during arthroscopic procedures. Despite the limited healing potential of chondral defects, several treatment options have been proposed. However, microfracture, osteochondral autograft (or allograft) transfer, autologous chondrocyte implantation, and matrix-induced autologous chondrocyte implantation are all associated with their respective shortcomings. As such, the optimal treatment for chondral defects of the knee remains unclear. Recently, many authors have advocated treating chondral defects with biological therapies and scaffold-based treatments. Bone marrow aspirate concentrate, a cell-based injection, has gained particular attention because of its differentiation capacity and potential role in tissue regeneration. In addition, scaffold cartilage treatments have emerged and reached clinical practice. BioCartilage is one form of scaffold, which consists of extracellular matrix, and has been claimed to promote the regeneration of hyaline-like cartilage. This article presents our technique of arthroscopic chondral defect repair using BMAC and BioCartilage.

Regeneration of hyaline cartilage in osteochondral lesion model using L-lysine magnetic nanoparticles labeled mesenchymal stem cells and their in vivo imaging.

Treatment of osteochondral defects continues to pose a major challenge for patients and orthopedic surgeons due to the limited healing potential of articular cartilage. Mesenchymal stem cells (MSCs) possess therapeutic potential for the treatment of osteochondral pain and pathology. However, it is necessary to use proper labeling and imaging agent of stem cells that can decipher its role posttransplantation. A major limitation of routinely used contrast agents is signal dilution over a period of time which limits its use for further studies. At the same time, regeneration of fibrocartilage over native hyaline cartilage also limits the use of conventional therapies. The present study evaluates the efficacy of bone marrow-derived mesenchymal stem cells (BMSCs) for the treatment of osteochondral defect in rats with the regeneration of hyaline cartilage in situ and in vivo monitoring of the stem cells using L-lysine functionalized magnetic iron oxide nanoparticles (lys-IONPs). L-lysine stabilizes the iron oxide nanoparticles, enhances the biocompatibility, and provides functionalities for efficient stem cell labeling. in vitro toxic effects of lys-IONPs on mitochondrial impairment, morphological alterations, and actin cytoskeleton reveal minimum damage to BM-MSCs. Histological data (H and E, Masson's trichrome and immunohistochemistry) describe the early initiation of healing and regeneration of hyaline-like cartilage over fibrocartilage in stem cell treated groups. MR scans demonstrate generation of hypointense signals in lys-IONPs-BMSCs with improved signal intensity and minimum loss over 28 days revealing its use as a long-term stem cell labeling and imaging agent.

Mesenchymal stem cell therapy combined with arthroscopic abrasion arthroplasty regenerates cartilage in patients with severe knee osteoarthritis: a case series.

Aim: To evaluate the safety and efficacy of adipose-derived mesenchymal stem cell (ADMSC) therapy in combination witharthroscopic abrasion arthroplasty (AAA) in advanced knee osteoarthritis (OA). Materials & methods: 27 patients with Grade IV OA of the knee underwent AAA and ADMSC therapy (50×106 ADMSCs at baseline and 6months). Clinical outcome was assessed over 36months. Structural change was determined using MRI. Results: Treatment was well tolerated with no serious adverse events. Clinically significant improvements in pain and function were observed. Reproducible hyaline-like cartilage regeneration was seen in all participants. Conclusion: ADMSC therapy combined with AAA in Grade IV OA results in reproducible pain, functional and structural improvements. This represents a joint preservation technique for patients with advanced OA of the knee. Trial registration number: ACTRN12617000638336.

Collagen Type I and Type II Expression Evaluation on Cartilage Defect Regeneration Treated with Dwikora–Ferdiansyah–Lesmono–Purwati (DFLP) Scaffold Supplemented with Adipose–Derived Stem Cells (ASCs) or Secretome: An In-Vivo Study

Abstract Cell-based therapies such as Scaffold, stem cells, and secretome, are one of the alternatives to enhance the regeneration of hyaline-like cartilage in cases of cartilage defects. This study is an in-vivo experiment using animal models, in which we apply a composite of DFLP (Dwikora-Ferdiansyah-Lesmono-Purwati) Scaffold and Adipose-Derived Stem Cells (ASCs) or Secretome to an injury model on the distal femoral trochlea of New Zealand White Rabbits. The animals were divided into four groups: (1) control (K); (2) Scaffold only (S); (3) Scaffold + ASCs (SA); (4) Scaffold + Secretome (SS). Animals were terminated in the 12th week, and an immunohistochemistry (IHC) evaluation for Collagen type I and II were done. Statistical analysis shows that collagen type I IHC between groups shows no significant difference (p = 0.546). Collagen type II IHC shows significant difference between groups (p = 0,016). The findings in this study showed that Scaffold + ASCs group and Scaffold + Secretome have better collagen type II expression compared to the control group. DFLP Scaffold composite with ASCs or Secretome shows potential for cartilage regeneration therapy by increasing type II collagen expression as in hyaline-like cartilage which may be used for regenerative therapy for cartilage defects. Keywords : DFLP Scaffold; Adipose-Derived Stem Cells (ASCs); Secretome; Collagen Type I; Collagen Type IICorrespondence : ianperbowo@me.com

Macroscopic and histologic evaluation of cartilage regeneration treated using xenogenic biodegradable porous sponge cartilage scaffold composite supplemented with allogenic adipose derived mesenchymal stem cells (ASCs) and secretome: An in vivo experimental study.

Articular cartilage is an avascular, alymphatic, and anisotropic tissue, these characteristics cause significant healing problems to injuries to the cartilage tissue. To overcome this problem, various techniques have been developed and widely used, but the cost-effectiveness and resulting tissue regeneration have never achieved hyaline-like cartilage that has the best biomechanical properties. The idea of this experiment is to use a Biodegradable Porous Sponge Cartilage (BPSC) Scaffold to enhance the regeneration of hyaline-like cartilage combined with microfracture technique and Adipose Derived Stem Cells (ASCs) or secretome on an animal model. A model defect was made on the femoral trochlea of a New Zealand white rabbit. Four groups were made to compare different treatment methods for osteochondral defects. The groups were: (1) Control group; (2) Scaffold Group; (3) Scaffold + ASCs Group; (4) Scaffold + Secretome Group. After 12 weeks, we terminate the animal models, then a macroscopic evaluation using the International Cartilage Research Society (ICRS) scoring system and Oswestry Arthroscopy Score (OAS) was done, followed by sectioning the specimen for microscopic evaluation using the O'Driscoll scoring system. The mean score for all treatment group were better compared to the control group grossly and histologically. The best mean score for macroscopic and microscopic evaluation was the group given Scaffold + ASCs. The application of BPSC scaffold enhances cartilage regeneration in larger osteochondral defects. Furthermore, the addition of ASCs or secretome along with the scaffold implantation further enhances the cartilage regeneration, in which ASCs shows better results.

Evaluation of autologous adipose-derived mesenchymal stem cell therapy in focal chondral defects of the knee: a pilot case series.

Aim: To evaluate the safety, pain, functional and structural improvements after autologous adipose-derived mesenchymal stem cell (ADMSC) therapy in combination with arthroscopic abrasion arthroplasty in focal chondral defects of the knee. Methods: Eight patients with a focal full thickness chondral defect of the knee underwent arthroscopic abrasion arthroplasty followed by postoperative intra-articular injections of autologous ADMSCs (50×106 ADMSCs at baseline and 6 months). Clinical outcome was assessed using numeric pain rating scale, Knee Injury and Osteoarthritis Outcome Score and the Western Ontario and McMaster Universities Osteoarthritis Index. Structural outcome was determined bymagnetic resonance imaging. Outcome was assessed over 24 months. Results: No serious adverse events occurred. Participants observed clinically significant improvement in pain and function. Magnetic resonance imaging analysis showed cartilage regeneration with T2 mapping values comparable to hyaline cartilage. Conclusion: Arthroscopic abrasion arthroplasty in combination with intra-articular ADMSC therapy results in reproducible pain, functional and structural improvements with regeneration of hyaline-like cartilage. Trial registration number: ACTRN12617000638336.

Synthesis and biological evaluation of a bioinspired, tissue-adhesive gellan gum-based hydrogel designed for minimally invasive delivery and retention of chondrogenic cells.

A dopamine-modified, bioinspired gellan gum hydrogel (STM-148B) with improved physicochemical and biological characteristics, suitable for minimally invasive cell delivery and retention in the context of cartilage repair, is herein presented. STM-148B's putative game-changing design characteristics include a highly biocompatible, animal-free and chemically defined composition, reproducibility of manufacture and ease of formulation. STM-148B undergoes rapid ionic crossinking by physiologically relevant mono and divalent cations to form stable 3D hydrogels that possess excellent tissue adhesiveness, such that additional fixation aids are rendered superfluous. STM-148B hydrogels maintain viability of mammalian cells and further promote up-regulation of the expression of healthy chondrogenic extracellular matrix markers upon stimulation. STM-148B is currently undergoing pre-clinical safety and efficacy assessment as a medical device for cell delivery and retention focussing on regeneration of hyaline-like cartilage and may represent a valuable addition to the armamentarium of tissue-engineering therapies for treatment of focal cartilage lesions.

Arthroscopic gel-type autologous chondrocyte implantation presents histologic evidence of regenerating hyaline-like cartilage in the knee with articular cartilage defect.

To investigate the clinical, radiological, and histological results of arthroscopic gel-type autologous chondrocyte implantation (GACI) in treating chondral defects of the knee. This study prospectively examined five males and five females with a mean age of 40.3 ± 10.3years who underwent arthroscopic GACI between March 2012 and February 2013. The gel comprised a mixture of 1ml of fibrinogen plus 0.1-0.2ml of thrombin. The mean size of chondral defect was 2.9 ± 1.2cm2 (range 1.2-5.4cm2). International knee documentation committee (IKDC) subjective score, knee injury and osteoarthritis outcome score (KOOS), knee society score, and visual analog scale (VAS) for pain were assessed preoperatively and during regular follow-up examinations performed for up to 5years postoperatively. Serial magnetic resonance imaging was performed for up to 2years after the surgery to observe healing, using the modified magnetic resonance observation of cartilage repair tissue (MOCART) score. In eight patients, second-look arthroscopy was performed at 1year after the implantation to assess the status of treated cartilage, and a portion of regenerated cartilage was harvested for histologic evaluation. The mean VAS score (p = 0.045), IKDC subjective score (p = 0.041), KOOS pain (p = 0.025), KOOS activities of daily living (p = 0.048), and KOOS quality of life (p = 0.029) showed significant improvement at 5years after the surgery. The modified MOCART evaluation showed that the scores were 59.5 ± 29.4 and 85.0 ± 8.0 at 12weeks and 2years after the operation, respectively. Histologic examination demonstrated a mean regenerated cartilage thickness of 3.5 ± 0.8mm and a mean Oswestry score of 8.2 ± 1.8. Immunohistochemistry analysis showed that the expression of collagen type II was more evident and more evenly distributed than collagen type I in regenerated cartilage. There was a significant correlation between Oswestry score and change in VAS scale from postoperative 2-5years. Arthroscopic GACI produces satisfactory clinical and radiologic outcomes, and histologic evaluation confirms sufficient regeneration of hyaline-like cartilage that correlates with improved symptoms. Therefore, it is an acceptable, minimally invasive, and technically simple option for the restoration of cartilage defects of the knee. IV.

Functionalized Scaffold for in Situ Efficient Gene Transfection of Mesenchymal Stem Cells Spheroids toward Chondrogenesis.

Multicellular mesenchymal stem cell (MSC) spheroids possess enhanced chondrogenesis ability and limited fibrosis, exhibiting advantage toward hyaline-like cartilage regeneration. However, because of the limited cell surfaces in spheroid exposed to DNA/vector, it is difficult to realize efficient gene transfection, most of which highly rely on cell-substrate interaction. Here, we report a poly(l-glutamic acid)-based porous scaffold with tunable inner surfaces that can sequentially realize cell-scaffold attachment and detachment, as well as the followed in situ spheroid formation. The attachment and detachment of cells from scaffold is achieved by the capture and release of fibronectin (Fn) via reversible imine linkage between aromatic aldehyde groups of scaffold and amino groups of Fn. Together with N, N, N-trimethyl chitosan chloride condensing plasmid DNA encoding transforming growth factor-β1 (pDNA-TGF-β1), cell attachment realizes efficient surface-mediated gene transfection. Conversion of scaffold stiffness can affect the adhesion shape of cells. Stiffer scaffold reinforces the adhesion, leading to the amplification of peripheral focal adhesions and the promotion of cell spreading, as well as the promotion of gene transfection efficiency. After cellular detachment from the scaffold via lysine treatment, the subsequent spheroid formation with extensive cell-cell interaction up-regulates the corresponding protein expression with a prolonged term. With the induction effect of the expressed TGF-β1, significantly enhanced chondrogenesis of MSCs in spheroids is achieved at 10 d in vitro. Well-regenerated cartilage at 8 weeks in vivo indicates that the present gene transfection system is a platform that can be potentially applied toward cartilage tissue engineering.

Component effect of stem cell-loaded thermosensitive polypeptide hydrogels on cartilage repair

Biophysical properties of the desired biomimetic scaffolds, such as porosity and elasticity, have been proven associated with the efficacy of cartilage regeneration. In this work, the copolymers of poly(l-alanine)-block-poly(ethylene glycol)-block-poly(l-alanine) (PA-PEG-PA) and poly(l-alanine-co-l-phenylalanine)-block-poly(ethylene glycol)-block-poly(l-alanine-co-l-phenylalanine) (PAF-PEG-PAF) with different ratios of alanine to phenylalanine were synthesized. The introduction of a hydrophobic amino acid, i.e., phenylalanine, into polyalanine-based thermosensitive hydrogel led to the enhanced gelation behaviors and upregulated mechanical properties. Moreover, the increase of phenylalanine content resulted in the enlarged pore size and enhanced mechanical strength of PAF-PEG-PAF thermogel, followed by the regeneration of hyaline-like cartilage with reduced fibrous tissue formation in vivo. The findings indicated the great potential of thermosensitive polypeptide hydrogels in cartilage tissue engineering. Statement of SignificanceArticular cartilage defect has limited self-repair ability due to the lack of blood supply and innervation, which may lead to knee osteoarthritis afterwards. Injectable hydrogels are demonstrated possessing outstanding properties as biomimetic scaffolds in cartilage tissue engineering, while the effect of biophysical properties on the efficacy of cartilage regeneration has not been revealed. Herein, the poly(ethylene glycol)-polypeptide triblock copolymers with different ratios of alanine to phenylalanine were synthesized. The sol-to-gel transition temperature and the critical gelation concentration decreased as the increased amount of phenylalanine unit, resulting in the enlarged pore size and enhanced mechanical strength. These features lead to better regeneration of hyaline-like cartilage with reduced fibrous tissue formation, indicating great potential of thermosensitive polypeptide hydrogels for efficient cartilage repair.

Characterization of polydactyly-derived chondrocyte sheets versus adult chondrocyte sheets for articular cartilage repair

BackgroundWe previously conducted a first-in-human clinical study of articular cartilage repair using autologous chondrocyte sheets and confirmed the regeneration of hyaline-like cartilage in all eight patients. However, regenerative medicine with autologous chondrocyte sheets requires the harvesting of tissue from healthy regions, and the quality of this tissue varies between individuals. To overcome such limitations, allogeneic transplantation is a promising treatment method, particularly for articular cartilage repair. In this study, we investigated the characteristics of polydactyly-derived chondrocyte sheets fabricated from the chondrocytes of young polydactyly donors.MethodsPolydactyly-derived chondrocyte (PD) sheets were fabricated from the tissue obtained from eight polydactyly donors (average age = 13.4 months). To create these PD sheets, chondrocytes at passage 2 or 3 were seeded on temperature-responsive culture inserts and cultured for 2 weeks. For comparison, adult chondrocyte sheets were fabricated from tissue obtained from 11 patients who underwent total knee arthroplasty (TKA; average age = 74 years). To create these TKA sheets, chondrocytes and synovial cells were cocultured, and the chondrocyte sheets were triple-layered according to the protocol from our previous clinical study. Cell count, cell viability, cell surface markers, cell histology, and humoral factors secreted by the sheets were characterized and compared between the PD sheets and TKA sheets.ResultsPolydactyly-derived chondrocytes proliferated rapidly to establish a layered structure with sufficient extracellular matrix and formed sheets that could be easily manipulated without tearing. Similar to TKA sheets, PD sheets expressed aggrecan and fibronectin at the protein level and the surface markers CD44, CD81, and CD90, which are characteristic of mesenchymal cells. PD sheets also produced significantly higher levels of transforming growth factor beta-1 and lower levels of matrix metalloproteinase-3 than those produced by TKA sheets, suggesting that young polydactyly-derived chondrocytes have advantages as a potential cell source.ConclusionsPD sheets exhibited characteristics thought to be important to chondrocyte sheets as well as proliferative capacity that may facilitate provision of a stable supply in the future.

Regeneration of hyaline-like cartilage in situ with SOX9 stimulation of bone marrow-derived mesenchymal stem cells.

Microfracture, a common procedure for treatment of cartilage injury, induces fibrocartilage repair by recruiting bone marrow derived mesenchymal stem cells (MSC) to the site of cartilage injury. However, fibrocartilage is inferior biomechanically to hyaline cartilage. SRY-type high-mobility group box-9 (SOX9) is a master regulator of chondrogenesis by promoting proliferation and differentiation of MSC into chondrocytes. In this study we aimed to test the therapeutic potential of cell penetrating recombinant SOX9 protein in regeneration of hyaline cartilage in situ at the site of cartilage injury. We generated a recombinant SOX9 protein which was fused with super positively charged green fluorescence protein (GFP) (scSOX9) to facilitate cell penetration. scSOX9 was able to induce chondrogenesis of bone marrow derived MSC in vitro. In a rabbit cartilage injury model, scSOX9 in combination with microfracture significantly improved quality of repaired cartilage as shown by macroscopic appearance. Histological analysis revealed that the reparative tissue induced by microfracture with scSOX9 had features of hyaline cartilage; and collagen type II to type I ratio was similar to that in normal cartilage. This short term in vivo study demonstrated that when administered at the site of microfracture, scSOX9 was able to induce reparative tissue with features of hyaline cartilage.