Potential of secretome of human fetal cartilage progenitor cells as disease modifying agent for osteoarthritis.

AIMS: Osteoarthritis (OA) is caused by an imbalance in the synthesis and degradation of cartilage tissue by chondrocytes. Therefore, a therapeutic agent for OA patients that can positively affect both synthesis and degradation is needed. However, current nonsurgical treatments for OA can barely achieve satisfactory long-term outcomes in cartilage repair. Human fetal cartilage progenitor cells-secretome (ShFCPC) has shown potent anti-inflammatory and tissue-repair effects; however, its underlying mechanisms and effects on OA have rarely been systematically elucidated. This study aims to analyze and evaluate the potency of ShFCPC in modifying OA process. MAIN METHODS: Herein, secreted proteins enriched in ShFCPC have been characterized, and their biological functions both in vitro and in vivo in an OA model are compared with those of human bone marrow-derived mesenchymal stem cells-secretome (ShBMSC) and hyaluronan (HA). KEY FINDINGS: Secretome analysis has shown that ShFCPC is significantly enriched with extracellular matrix molecules involved in many effects of cellular processes required for homeostasis during OA progression. Biological validation in vitro has shown that ShFCPC protects chondrocyte apoptosis by suppressing the expression of inflammatory mediators and matrix-degrading proteases and promotes the secretion of pro-chondrogenic cytokines in lipopolysaccharide-induced coculture of human chondrocytes and SW982 synovial cells compared with ShBMSC. Moreover, in a rat OA model, ShFCPC protects articular cartilage by reducing inflammatory cell infiltration and M1/M2 macrophage ratio in the synovium, which directly contributes to an increase in immunomodulatory atmosphere and enhances cartilage repair compared to ShBMSC and HA. SIGNIFICANCE: Our findings support clinical translations of ShFCPC as a novel agent for modifying OA process.

Conditioned media derived from human fetal progenitor cells improves skin regeneration in burn wound healing.

Stem cells are known to have excellent regenerative ability, which is primarily facilitated by indirect paracrine factors, rather than via direct cell replacement. The regenerative process is mediated by the release of extracellular matrix molecules, cytokines, and growth factors, which are also present in the media during cultivation. Herein, we aimed to demonstrate the functionality of key factors and mechanisms in skin regeneration through the analysis of conditioned media derived from fetal stem cells. A series of processes, including 3D pellet cultures, filtration and lyophilization is developed to fabricate human fetal cartilage-derived progenitor cells-conditioned media (hFCPCs-CM) and its useful properties are compared with those of human bone marrow-derived MSCs-conditioned media (hBMSCs-CM) in terms of biochemical characterization, and in vitro studies of fibroblast behavior, macrophage polarization, and burn wound healing. The hFCPCs-CM show to be devoid of cellular components but to contain large amounts of total protein, collagen, glycosaminoglycans, and growth factors, including IGFBP-2, IGFBP-6, HGF, VEGF, TGF ß3, and M-CSF, and contain a specific protein, collagen alpha-1(XIV) compare with hBMSCs-CM. The therapeutic potential of hFCPCs-CM observes to be better than that of hBMSCs-CM in the viability, proliferation, and migration of fibroblasts, and M2 macrophage polarization in vitro, and efficient acceleration of wound healing and minimization of scar formation in third-degree burn wounds in a rat model. The current study shows the potential therapeutic effect of hFCPCs and provides a rationale for using the secretome released from fetal progenitor cells to promote the regeneration of skin tissues, both quantitatively and qualitatively. The ready-to-use product of human fetal cartilage-derived progenitor cells-conditioned media (hFCPCs-CM) are fabricated via a series of techniques, including a 3D culture of hFCPCs, filtration using a 3.5 kDa cutoff dialysis membrane, and lyophilization of the CM. hFCPCs-CM contains many ECM molecules and biomolecules that improves wound healing through efficient acceleration of M2 macrophage polarization and reduction of scar formation.

Fabrication of decellularized meniscus extracellular matrix according to inner cartilaginous, middle transitional, and outer fibrous zones result in zone-specific protein expression useful for precise replication of meniscus zones.

Meniscus is a fibrocartilage composite tissue with three different microstructual zones, inner fibrocartilage, middle transitional, and outer fibrous zone. We hypothesized that decellularized meniscus extracellular matrix (DMECM) would have different characteristics according to zone of origin. We aimed to compare zone-specific DMECM in terms of biochemical characteristics and cellular interactions associated with tissue engineering. Micronized DMECM was fabricated from porcine meniscus divided into three microstructural zones. Characterization of DMECM was done by biochemical and proteomic analysis. Inner DMECM showed the highest glycosaminoglycan content, while middle DMECM showed the highest collagen content among groups. Proteomic analysis showed significant differences among DMECM groups. Inner DMECM showed better adhesion and migration potential to meniscus cells compared to other groups. DMECM resulted in expression of zone-specific differentiation markers when co-cultured with synovial mesenchymal stem cells (SMSCs). SMSCs combined with inner DMECM showed the highest glycosaminoglycan in vivo. Outer DMECM constructs, on the other hand, showed more fibrous tissue features, while middle DMECM constructs showed both inner and outer zone characteristics. In conclusion, DMECM showed different characteristics according to microstructural zones, and such material may be useful for zone-specific tissue engineering of meniscus.

Corneal Repair with Adhesive Cell Sheets of Fetal Cartilage-Derived Stem Cells.

BACKGROUND: Corneal scarring or disease may lead to severe corneal opacification and consequently, severe loss of vision due to the complete loss of corneal epithelial cells. We studied the use of epithelial cell sheets differentiated from fetal cartilage-derived stem cells (FCSC) to resurface damaged cornea. METHODS: The FCSC were isolated from the femoral head of immature cartilage tissue. The ability of the FCSCs to differentiate into corneal epithelial cells was evaluated using differentiation media at 2 days and 7 days post-seeding. A sheet fabricated of FCSCs was also used for the differentiation assay. The results of the in vitro studies were evaluated by immunocytochemistry and Western blots for corneal epithelial cell markers (CK3/12 and Pax6) and limbal epithelial stem cell markers (ABCG2 and p63). To test the material in vivo, an FCSC-sheet was applied as a treatment in a chemically burned rabbit model. The healing ability was observed histologically one week after treatment. RESULTS: The in vitro experiments showed morphological changes in the FCSCs at two and seven days of culture. The differentiated cells from the FCSCs or the FCSC-sheet expressed corneal epithelial cells markers. FCSC were create cell sheet that successfully differentiated into corneal epithelial cells and had sufficient adhesion so that it could be fused to host tissue after suture to the ocular surface with silk suture. The implanted cell sheet maintained its transparency and the cells were alive a week after implantation. CONCLUSION: These results suggest that carrier-free sheets fabricated of FCSCs have the potential to repair damaged corneal surfaces.

Decellularized Porcine Epiphyseal Plate-Derived Extracellular Matrix Powder: Synthesis and Characterization.

The aim of this study was to develop a porcine epiphyseal plate-derived extracellular matrix powder (PEPEP) for epiphyseal plate regeneration. PEPEP was characterized by chemical assay to determine the contents of DNA and epiphyseal plate complex chemical components (glycosaminoglycan and hydroxyproline). The effects of PEPEP on the viability, proliferation, and differentiation of human bone marrow mesenchymal stem cells (hBMSCs) were also evaluated. hBMSCs cultured in PEPEP exhibited a good distribution with excellent viability after 72 h, demonstrating the ability of PEPEP to support hBMSC proliferation. At week 4 and 6 in vitro, the PEPEP + hBMSCs structure showed chondrogenic ability and an increase in expression of collagen type I, type II, and type X. PEPEP showed a promising ability to enhance cartilage formation and promote chondrocyte differentiation, maturation, and hypertrophy. The results provide insights into the feasibility of PEPEP as a potential material for tissue engineering applications.

Engineered cartilage utilizing fetal cartilage-derived progenitor cells for cartilage repair.

The aim of this study was to develop a fetal cartilage-derived progenitor cell (FCPC) based cartilage gel through self-assembly for cartilage repair surgery, with clinically useful properties including adhesiveness, plasticity, and continued chondrogenic remodeling after transplantation. Characterization of the gels according to in vitro self-assembly period resulted in increased chondrogenic features over time. Adhesion strength of the cartilage gels were significantly higher compared to alginate gel, with the 2-wk group showing a near 20-fold higher strength (1.8 ± 0.15 kPa vs. 0.09 ± 0.01 kPa, p < 0.001). The in vivo remodeling process analysis of the 2 wk cultured gels showed increased cartilage repair characteristics and stiffness over time, with higher integration-failure stress compared to osteochondral autograft controls at 4 weeks (p < 0.01). In the nonhuman primate investigation, cartilage repair scores were significantly better in the gel group compared to defects alone after 24 weeks (p < 0.001). Cell distribution analysis at 24 weeks showed that human cells remained within the transplanted defects only. A self-assembled, FCPC-based cartilage gel showed chondrogenic repair potential as well as adhesive properties, beneficial for cartilage repair.

Cross-linked cartilage acellular matrix film decreases postsurgical peritendinous adhesions.

Cartilage extracellular matrix contains antiadhesive and antiangiogenic molecules such as chondromodulin-1, thrombospondin-1, and endostatin. We have aimed to develop a cross-linked cartilage acellular matrix (CAM) barrier for peritendinous adhesion prevention. CAM film was fabricated using decellularized porcine cartilage tissue powder and chemical cross-linking. Biochemical analysis of the film showed retention of collagen and glycosaminoglycans after the fabrication process. Physical characterization of the film showed denser collagen microstructure, increased water contact angle, and higher tensile strength after cross-linking. The degradation time in vivo was 14 d after cross-linking. The film extract and film surface showed similar cell proliferation, while inhibiting cell migration and cell adhesion compared to standard media and culture plate, respectively. Application of the film after repair resulted in similar tendon healing and significantly less peritendinous adhesions in a rabbit Achilles tendon injury model compared to repair only group, demonstrated by histology, ultrasonography, and biomechanical testing. In conclusion, the current study developed a CAM film having biological properties of antiadhesion, together with biomechanical properties and degradation profile suitable for prevention of peritendinous adhesions.

A dual synergistic of curcumin and gelatin on thermal-responsive hydrogel based on Chitosan-P123 in wound healing application.

This study aimed to fabricate the potential therapeutic scaffold to efficiently and safely fastening skin wound healing. A biocompatible grafting polymer-based thermal sensitive hybrid hydrogel (Chitosan-P123, CP) containing gelatin and curcumin was designed to be suitable stiffness for tissue regeneration. A detailed in the rheological study found that the encapsulated agents induced the change in the stiffness of the hydrogel from the hard to the soft. Especial, the thermally induced phase transition of CP hydrogel was governed by the participant of gelatin rather than curcumin. For example, at 25 wt% gelatin, CP hydrogel exhibited a unique gel-sol-gel transition following the function of temperature. Moreover, in vitro investigation revealed that the hybrid hydrogel provides the capacity of especially induced curcumin release with a sustainable rate as well as the excellent biocompatibility scaffold. Altogether with in vivo study, the hybrid hydrogel highlighted the advance of the dual synergistic of curcumin and gelatin in development of smart scaffold system, which promoted the efficacy in the regeneration of the structure and the barrier's function of damaged skin such as wound or skin cancer.

Fabrication and characterization of 3D-printed elastic auricular scaffolds: A pilot study.

OBJECTIVE: Aesthetic reconstruction of the external ear is challenging due to the complex anatomical shape of the auricle. Recently, artificial scaffolds such as Medpor (Stryker, Kalamasoo, MI, USA) have become widely used in ear reconstruction. However, the Medpor scaffold is stiffer than the natural ear, which may lead to discomfort, and moreover has uniform design for every patient. In this study, we investigated whether three-dimensional (3D)-printed artificial polyurethane (PU) scaffolds are suitable for auricular reconstruction. METHODS: PU scaffolds were fabricated using 3D printing according to a design derived from a digital imaging and communications in medicine (DICOM) image of the human auricle. The microstructure of the scaffolds was observed using scanning electron microscopy, and the porosity was examined. Cell proliferation on the scaffolds was assessed in vitro using tonsil-derived mesenchymal stem cells to evaluate the biocompatibility of the scaffolds. The scaffolds were implanted in C57BL/6 mice, and histological analysis was performed. RESULTS: The structural study revealed that the 3D-printed porous PU scaffolds have rectangular microstructure with regular pitch and line, as well as high porosity (56.46% ± 10.22%) with a pore diameter of 200 µm. The mechanical properties of the 3D-printed PU scaffolds were similar to those of the human auricle cartilage. Cell proliferation on the PU scaffolds was greater than that on Medpor scaffolds. Histological evaluation demonstrated that the porous parts of the PU scaffolds became filled with collagen and vascular tissue. CONCLUSION: Elastic, porous PU scaffolds can be obtained using 3D printing, have biomechanical properties similar to those of the natural ear, and are suitable for use in auricular reconstruction. LEVEL OF EVIDENCE: NA Laryngoscope, 129:351-357, 2019.

Sizable Scaffold-Free Tissue-Engineered Articular Cartilage Construct for Cartilage Defect Repair.

This study introduces an implantable scaffold-free cartilage tissue construct (SF) that is composed of chondrocytes and their self-produced extracellular matrix (ECM). Chondrocytes were grown in vitro for up to 5 weeks and subjected to various assays at different time points (1, 7, 21, and 35 days). For in vivo implantation, full-thickness defects (n = 5) were manually created on the trochlear groove of the both knees of rabbits (16-week old) and 3 week-cultured SF construct was implanted as an allograft for a month. The left knee defects were implanted with 1, 7, and 21 days in vitro cultured scaffold-free engineered cartilages. (group 2, 3, and 4, respectively). The maturity of the engineered cartilages was evaluated by histological, chemical and mechanical assays. The repair of damaged cartilages was also evaluated by gross images and histological observations at 4, 8, and 12 weeks postsurgery. Although defect of groups 1, 2, and 3 were repaired with fibrocartilage tissues, group 4 (21 days) showed hyaline cartilage in the histological observation. In particular, mature matrix and columnar organization of chondrocytes and highly expressed type II collagen were observed only in 21 days in vitro cultured SF cartilage (group 4) at 12 weeks. As a conclusion, cartilage repair with maturation was recapitulated when implanted the 21 day in vitro cultured scaffold-free engineered cartilage. When implanting tissue-engineered cartilage, the maturity of the cartilage tissue along with the cultivation period can affect the cartilage repair.

Development and Efficacy Testing of a "Hollow Awl" That Leads to Patent Bone Marrow Channels and Greater Mesenchymal Stem Cell Mobilization During Bone Marrow Stimulation Cartilage Repair Surgery.

PURPOSE: To investigate whether microfracture with a cannulated hollow awl can yield more patent marrow channels and allow greater mobilization of the reparable cells to the defect compared to the conventional awl with blunt end in human knee joints. METHODS: Patients who were planned for 1-stage bilateral total knee arthroplasty due to degenerative osteoarthritis with well-preserved lateral femoral condylar cartilage were retrospectively included. A 10-mm × 20-mm, rectangular, full-thickness chondral defect was made on the lateral femoral condyle on each knee joint. The 6-holed microfracture procedure, each at 9 mm depth and 3 mm separation of perforations, was followed using a hollow awl in one knee and using a conventional awl in the other knee, respectively. The bleeding through the microfracture holes was observed and collected using an absorbable gelatin sponge and was analyzed microscopically by colony forming unit-fibroblast assays and automated cell counting method. The representative 3 bony samples of the distal lateral femoral condyles obtained were also scanned with micro-computed tomography (micro-CT) for morphometric analysis (percent bone volume, trabecular separation, and total porosity) of subchondral bone microarchitecture of the microfracture holes. RESULTS: Twenty-two patients were enrolled, and the mean age was 70.8 ± 6.1 (58-83) years. Compared with the conventional awl group, the hollow awl group had a significantly greater amount of bleeding (1.8 ± 0.2 g vs 1.1 ± 0.1 g; P < .001) and a greater number of mesenchymal stem cells in the blood clot (21,474.0 ± 3,911.1 vs 13,329.7 ± 3,311.0; P = .004). The hollow awl group also showed overall more patent marrow channels around the adjacent subchondral bone of the microfracture hole, with greater trabecular separation on micro-CT analysis (P < .001). CONCLUSIONS: Compared to the conventional awl, microfracture with a cannulated hollow awl can yield more patent marrow channels at the adjacent subchondral bone of the microfracture hole and result in greater mobilization of the reparable cells to the defect in human knee joints. LEVEL OF EVIDENCE: Level III, therapeutic case control.

Histomorphochemical comparison of microfracture as a first-line and a salvage procedure: is microfracture still a viable option for knee cartilage repair in a salvage situation?

Microfracture is considered as the first-line procedure for knee cartilage repair, but the results of microfracture seem less predictable and rather controversial in a salvage situation. Thus, the purpose of the study was to histomorphochemically compare microfracture as a salvage procedure with microfracture as a first-line procedure in a rabbit model. We hypothesized that microfracture in a salvage situation would result in histomorphochemically inferior cartilage repair compared to microfracture as a first-line procedure, and the inferiority would be attributed to less migration of reparable marrow cells to the defect due to destruction of microarchitecture of the subchondral bone. Thirty-six New Zealand white rabbits were divided into three groups: (i) untreated full-thickness chondral defect, (ii) single microfracture treatment (first microfracture group), and (iii) repeated microfracture in 8 weeks after the first procedure (second microfracture group). In each group, rabbits were sacrificed at the end of 8 weeks, and osteochondral specimens at the repair sites were obtained for histomorphochemical analysis. Results showed that microfracture as a salvage procedure resulted in overall inferior cartilage repair histomorphochemically compared with microfracture as a first-line procedure, which correlated with deteriorative changes in the quality of underlying subchondral bone rather than intrinsic incapability to recruit the reparative cells in the defect area. In conclusion, although a comparable number of reparable cells and a mechanically weakened subchondral bone are anticipated, more study is necessary to clearly determine when a microfracture should be performed in a situation.