Cartilage Particles Research Articles

The iPSC secretome is beneficial for in vitro propagation of primary osteoarthritic chondrocytes cell lines

BackgroundOne of the obstacles to autologous chondrocyte implantation (ACI) is obtaining a large quantity of chondrocytes without depletion of their properties. The conditioned medium (CM) from different subpopulations of stem cells (mesenchymal stromal cells (MSC) or induced pluripotent stem cells (iPSC)) could be a gamechanger. MSCs' potential is related to the donor's health and age, which could be omitted when, as a source, iPSCs are used. There is a lack of data regarding their use in the chondrocyte culture expansion. Thus, we wanted to verify whether iPSC-CM could be beneficial for the cell culture of primary chondrocyte cells. MethodsWe added the iPSC-CMs from GPCCi001-A and ND 41658*H cells to the culture of primary chondrocyte cell lines isolated from OA patients (n = 6) for other two passages. The composition of the CM was evaluated using Luminex technology. Then, we analysed the senescence, proliferation rate and using flow cytometry: viability, distribution of cell cycle phases, production of reactive oxygen species (ROS) and double-strand breaks. The cartilage-related markers were evaluated using Western blot and immunofluorescence. Additionally, a three-dimensional cell culture was used to determine the potential to form cartilage particles. ResultsiPSC-CM increased proliferation and diminished cell ROS production and senescence. CM influenced the cartilage-related protein expression and promoted the growth of cartilage particles. The cell exposed to CM did not lose the ECM proteins, suggesting the chondroprotective effect for prolonged culture time. ConclusionOur preliminary results suggest a beneficial effect on maintaining chondrocyte biology during in vitro expansion.

183. Customizable, Biocompatible And Low-cost 3D Printed Implants For Craniofacial Reconstruction

PURPOSE: Craniofacial reconstruction or enhancement requires autologous, cadaveric or alloplastic implants, often requiring intraoperative modification to optimize shape and fit for a particular patient, introducing associated risks of infection, extrusion, expense and donor site morbidity. Given this, there is substantial need for biocompatible, customizable and low-cost facial implants. This study examines the biocompatibility and durability of various polylactic acid (PLA) implant designs +/- decellularized cartilage infill to assess translatability to craniofacial reconstruction. METHODS: Cartilage harvested from ovine ribs was minced or zested and decellularized. Using 3D modeling software, dorsal nasal scaffolds were custom-designed with contours similar to commercially available silicone implants. Scaffolds were printed in polylactic acid (PLA) on a PRUSA i3 MK3S+ printer and implanted subcutaneously on rat dorsa. Two scaffolds were designed as external “cages” of low or high porosity and were without internal supports. These cages were implanted empty or with either minced or zested decellularized cartilage infill, yielding 6 total treatment groups, n=4 each. The remaining two scaffolds had internal PLA filament supports and were implanted without decellularized cartilage infill. Scaffolds were explanted 3 and 6 months after implantation, after which they underwent same-day volumetric analysis via microCT, then Formalin-fixed and embedded in paraffin and sectioned at 10μm for histopathologic analysis. RESULTS: Volumetric analysis of explants at 3 and 6 months revealed retention of implant contours and volume across rebar and minced cartilage-filled scaffolds. Tissue ingrowth between cartilage particles was evident between 3 and 6 months on MicroCT. Empty and zested cartilage-filled cages displayed significant volume loss relative to volume at implant (p < 0.05) with explanted constructs grossly collapsed; intergroup comparison revealed rebar and minced cartilage-filled constructs had superior volume retention relative to empty and zested cartilage-filled cages (p < 0.05). Histopathology with hematoxylin and eosin and safranin-O stains at the 3 versus 6-month timepoints indicated resolving lymphocytic inflammatory response, increased tissue vascularization and improved collagen deposition. CONCLUSION: This study shows that PLA facial implants with sufficient internal supports are a feasible and safe alternative to current craniofacial reconstructive implant options. The biocompatibility of PLA and decellularized cartilage infill promotes ingrowth of healthy tissue with collagen deposition and neovascularization. Further, design and fabrication of these implants can be completed quickly and at minimal cost, allowing for affordable custom-printed implants with a minimal risk profile.

Decellularized extracellular matrix particle-based biomaterials for cartilage repair applications

Cartilage Decellularized ExtraCellular Matrix (dECM) materials have shown promising cartilage regeneration capacity due to their chondrogenic bioactivity. However, the limited retention of ECM components and the reduced integrity of functional ECM molecules during traditional decellularization processes impair the biomimicry of these materials. The current study aims to fabricate biomimetic materials containing decellularized cartilage particles that have an intact molecular structure and native composition as biomaterial inks and hydrogels for cartilage repair. For this, we established a novel two-fraction decellularization strategy for the preparation of reconstituted dECM (rdECM) particles by mixing the two-fraction components, as well as a one-fraction decellularization strategy for the preparation of biomimetic dECM (bdECM) particles. Hyaluronic acid-tyramine (THA) hydrogels containing rdECM or bdECM particles were produced and characterized via rheological test, swelling and stability evaluation, and compression test. The results showed that our novel decellularization strategies preserved intact proteoglycans and collagen at a higher retention rate with adequate DNA removal compared to traditional methods of decellularization. The addition of rdECM or bdECM particles significantly increased the shear moduli of the THA bioinks while preserving their shear-thinning properties. bdECM particle-embedded THA hydrogels also achieved long-term stability with a swelling ratio of 70% and high retention of glycosaminoglycans and collagen after long-term incubation, while rdECM particle-embedded THA hydrogels showed unsatisfactory stability as self-standing biomaterials. Compared to pure THA hydrogels, the addition of bdECM particles significantly enhanced the compression moduli. In summary, our decellularization methods are successful in the retention of functional and intact cartilage components with high yield. Both rdECM and bdECM particles can be supplemented in THA bioinks for biomimetic cartilage 3D printing. Hydrogels with cartilage bdECM particles possess the functional structure and the natural composition of cartilage ECM, long-term stability, and enhanced mechanical properties, and are promising biomaterials for cartilage repair.

Biocompatible and functional properties of a microdispersed tissue-specific 3D matrix from decellularized porcine cartilage

In contrast to decellularization of soft tissues for use as tissue-specific matrices in the creation of tissue-engineered constructs, decellularization of cartilage tissue requires several processing techniques, which can negatively affect the biocompatibility and functional properties of the native extracellular matrix (ECM).Objective: to study the biocompatible and functional properties of microdispersed tissue-specific 3D matrix from a porcine cartilage that is decellularized by sequential use of chemical, physical and enzymatic techniques.Materials and methods. For decellularization, microdispersed cartilage particles (MCPs), obtained by cryomilling, were incubated in detergent solutions (sodium dodecyl sulfate and Triton X-100), then treated with supercritical carbon dioxide (scCO2) with 10% ethanol and DNase I. The Ames test (Salmonella typhimurium reverse mutation assay) was used to determine the genotoxicity of decellularized microdispersed cartilage particles (dMCPs). Local and general toxic effects, as well as resorption of dMCPs were studied in vivo on sexually mature outbred rats. Decellularized MCP specimens (10 mg) were implanted into the thigh muscle tissue. Viability of human adipose-derived mesenchymal stem/stromal cells (hAdMSCs), when cultured on dMCPs, was analyzed by in vivo microscopy, stained with fluorescent Calcein AM dye. Cell metabolic activity was assessed using PrestoBlue™ Cell Viability Reagent.Results. It has been proven that porcine dMCPs implanted in rat muscle after treatment with scCO2 do not exhibit local and general toxic effects, and do not show genotoxicity and negative effects on the reproductive system of animals. After 6 months of in vivo experiment, most (87%) of the implanted decellularized cartilage was resorbed. It was shown that the resulting matrices are able to support adhesion and proliferation of hAdMSCs. Conclusion. Porcine dMCP specimens are suitable for biocompatible medical products in terms of local and general toxic effects, genotoxicity and reproductive toxicity, and can be used as a matrix for creating cell- and tissue-engineered cartilage constructs.

Effect of Mechanical Mincing on Minimally Manipulated Articular Cartilage for Surgical Transplantation

Background: Point-of-care treatment options for medium to large symptomatic articular cartilage defects are limited. Minced cartilage implantation is an encouraging single-stage option, providing fresh viable autologous tissue with minimal morbidity and cost. Purpose: To determine the histological properties of mechanically minced versus minimally manipulated articular cartilage. Study Design: Controlled laboratory study. Methods: Remnant articular cartilage was collected from fresh femoral condylar allografts. Cartilage samples were divided into 4 groups: cartilage explants with or without fibrin glue and mechanically minced cartilage with or without fibrin glue. Samples were cultured for 42 days. Chondrocyte viability was assessed using live/dead assay. Cellular migration and outgrowth were monitored using bright-field microscopy. Extracellular matrix deposition was assessed via histological staining. Proteoglycan content and synthesis were assessed using dimethylmethylene blue assay and radiolabeled 35S-sulfate, respectively. Type II collagen (COL2A1) gene expression was analyzed via polymerase chain reaction. Results: The mean viability of minced cartilage particles (34% ± 14%) was not significantly reduced compared with baseline (46% ± 13%) on day 0 (P = .90). After culture, no significant difference in the percentage of live cells was appreciated between mechanically minced (58% ± 23%) and explant (73% ± 14%) cartilage in the presence of fibrin glue (P = .52). The addition of fibrin glue did not significantly affect the viability of cartilage samples. The qualitative assessment revealed comparable cellular migration and outgrowth between groups. Proteoglycan synthesis was not significantly different between groups. Histological analysis findings were positive for COL2A1 in all groups, and matrix formation was appreciated in all groups. COL2A1 expression in minced cartilage (1.72 ± 1.88) was significantly higher than in explant cartilage (0.15 ± 0.07) in the presence of fibrin glue (P = .01). Conclusion: Mechanically minced articular cartilage remained viable after 42 days of culture in vitro and was comparable with cartilage explants with regard to cellular migration, outgrowth, and extracellular matrix synthesis. Clinical Relevance: Mechanically minced articular cartilage is an encouraging intervention for the treatment of symptomatic cartilage defects. Further translational work is warranted to determine the viability of minced cartilage implantation as a single-stage therapeutic intervention in vivo.

Supercritical carbon dioxide as a tool for improving the biocompatible properties of biopolymer and tissue specific scaffolds for tissue engineering

Objective: to investigate the efficacy of supercritical carbon dioxide (sc-CO2) for enhancштп the biocompatibility of biopolymer scaffolds from biodegradable materials and tissue-specific scaffolds from decellularized porcine liver slices (PLSs) or fine porcine cartilage particles (FPCPs).Materials and methods. Biopolymer scaffolds of a polyoxy(butyrate-co-valerate) and gelatin copolymer composition, 4 mm in diameter and 80 mm in length, were formed by electrospinning (NANON-01A, MECC CO, Japan) and stabilized by incubation in glutaraldehyde vapor for 48 hours at room temperature. For decellularization, PLSs and FPCPs were incubated under periodic stirring in buffer (pH = 7.4) solutions of sodium dodecyl sulfate (0.1%) and Triton X-100 with increasing concentrations (1, 2, and 3%). Treatment in a sc-CO2 atmosphere was done at 150–300 bar pressure, 35 °C temperature, and 0.25–2.5 mL/min flow rate of sc-CO2 for 8–24 hours. 10% ethanol was introduced as a polarity modifier. Cytotoxicity was studied according to GOST ISO 10993-5-2011. The growth of NIH/3T3 in the presence of samples was studied using an interactive optical system IncuCyte Zoom.Results. The effect of the sc-CO2 flow rate and pressure, and the effect of addition of ethanol, on the biocompatibility of scaffolds was investigated. It was found that treatment at a low sc-CO2 flow rate (0.25 mL/min) does not achieve the required cytotoxicity. Complete absence of cytotoxicity in biopolymer scaffolds was achieved in the presence of 10% ethanol, at a sc-CO2 flow rate of 2.5 mL/min, 300 bar pressure and 35 °C temperature after 8 hours of treatment. Effective removal of cytotoxic detergents from decellularized liver occurs already at a 150-bar pressure and does not require the addition of ethanol. Adding ethanol to sc-CO2 eliminates not only the cytotoxic, but also the cytostatic effect of tissue-specific scaffolds.Conclusion. Sc-CO2 treatment is an effective way to enhance the biocompatibility of three-dimensional porous matrices produced using cytotoxic substances: bifunctional crosslinking agents for biopolymer scaffolds and surfactants in the case of tissue-specific matrices. Addition of ethanol as a polarity modifier improves the treatment efficiency by eliminating both cytotoxic and cytostatic effects.

Cartilage Particles can Promote Chondrogenesis of Adipose-Derived Stromal Cells on Poly(ε-Caprolactone)/Fibrin Hybrid Constructs Prepared via Sandwich Model

Electrospun fibers have demonstrated a remarkable potential as a framework structure in the fabrication of cartilage tissue engineering (CTE) scaffolds. Various extracellular matrices have been incorporated into electrospun scaffolds to mimic and simulate the extracellular environment. The objective of this study was to fabricate hybrid constructs using composite electrospun scaffolds based on poly (ε-caprolactone) (PCL) and cartilage-derived matrix (CDM) and fibrin hydrogel to improve the viability and differentiation of human adipose-derived stromal cells (ADSCs) for CTE applications.Initially, PCL and PCL-CDM electrospun mats were fabricated. Fibrin/ ADSCs hydrogel were seeded on PCL- CDM mats and arranged layer-by-layer using sandwich technique. This method has been employed to increase cell seeding and infiltration efficiency through the entire mass of the scaffold. Real-time reverse-transcription polymerase chain reaction (RT- PCR), were performed to examine the expression of collagen types II and X, SOX9 and aggrecan. The production of glycosaminoglycan (GAG) was also tested in vitro by Toluidine blue stain and biochemical assay in the cultured scaffolds.The findings demonstrated that incorporation of CDM in PCL fibers results in improved cell viability. Hematoxylin and eosin staining showed that the sandwich method resulted in homogenous cell seeding within the scaffold. Overall, the RT- PCR, biochemical and histological results, showed that incorporation of the CDM into PCL/fibrin sandwich scaffolds stimulated ADSCs chondrogenesis and produced the products which increased expression of chondrogenic genes. It also, enhanced GAG synthesis compared to PCL/fibrin scaffolds.These findings suggest PCL-CDM/fibrin can be considered as an appropriate hybrid scaffold for CTE applications.

Human Amniotic Mesenchymal Stem Cell Sheets Encapsulating Cartilage Particles Facilitate Repair of Rabbit Osteochondral Defects

Background: Human amniotic mesenchymal stem cells (hAMSCs) are being widely applied in various fields. Therefore, hAMSCs represent a promising candidate to facilitate cartilage regeneration. Nonetheless, no studies have investigated the application of hAMSC sheets to repair cartilage defects in vivo. Purpose: To evaluate hAMSC sheets encapsulating cartilage particles to promote repair of rabbit osteochondral defects. Study Design: Controlled laboratory study. Methods: hAMSC sheets were constructed with passage 3 hAMSCs. The phenotypic and structural characteristics of hAMSC sheets were evaluated by flow cytometry and scanning electron microscopy, respectively. The potential for chondrogenic differentiation of hAMSC sheets was assessed by cartilage-specific marker staining, immunohistochemistry, and mRNA and protein expression (SOX9, COLII, and ACAN). Osteochondral defects (diameter, 3.5 mm; depth, 3 mm) were created in the left patellar grooves of 20 New Zealand White rabbits (female or male). The defects were treated with hAMSC sheet/cartilage particles (n = 5), cartilage particles (n = 5), hAMSC sheets (n = 5), or fibrin glue (n = 5). Macroscopic and histological evaluations of the regenerated tissue were conducted after 3 months. The survival time and differentiation of transplanted hAMSCs in the defect area were evaluated by immunofluorescence. Results: hAMSC sheets had a multilayered structure, with cells stacked layer by layer. Importantly, hAMSC sheets highly expressed phenotypic markers of mesenchymal stem cells. Cartilage-specific marker staining and immunohistochemistry were positive, and mRNA and protein expression was higher in the chondrogenically induced hAMSC sheet group than in the hAMSC sheet group (P < .05). hAMSC sheet/cartilage particles formed a large amount of hyaline-like cartilage in the defect area. In addition, macroscopic and histological scores were significantly higher than those in the other groups. Integration with surrounding normal cartilage and subchondral bone regeneration in the hAMSC sheet/cartilage particles group were better when compared with the other groups. A large number of human nuclear-specific antigen-positive cells were observed in the defect area of hAMSC sheet/cartilage particles and hAMSC sheet groups. Moreover, some positive cells expressed SOX9. Conclusion: hAMSC sheets encapsulating cartilage particles facilitate osteochondral defect repair. Clinical Relevance: Delivery of cells in the form of a cell sheet in conjunction with cartilage particles provides a novel approach for cell-based cartilage regeneration.

Получение микродисперсного тканеспецифического децеллюляризованного матрикса из суставного хряща свиньи

A method for obtaining a microdispersed tissue-specific matrix from decellularized porcine articular cartilage while maintaining morphological and functional properties of the extracellular matrix with no signs of cytotoxicity was developed. The cartilage particle size distribution in suspension after cryogenic grinding was determined using laser diffraction analysis. The range of sizes of the obtained cartilage microparticles suggests the possibility of their administration by injection (<220 μm). The combination of stages, including three freeze/thaw cycles (–196/37°C) followed by treatment with solutions of surface-active substances (surfactants), sodium dodecyl sulfate and Triton X-100, and DNase resulted in the complete removal of non-decellularized microparticles. The residual DNA content was 9.11 ± 1.13 ng/mg of tissue. The effectiveness of surfactant washing was assessed by the cytotoxicity of the matrix on the culture of human adipose derived mesenchymal stromal cells (hADSCs). To assess the hemocompatibility of the obtained samples, their hemolytic activity was studied in vitro. The adhesion and proliferation of hADSCs on the matrix surface were studied on day 21 of cultivation. The matrix did not exhibit hemolytic activity and cytotoxicity with respect to hADSCs. hADSCs in the samples were actively proliferating on the matrix surface. The biocompatibility and hemocompatibility of the obtained matrix in vitro indicate its potential for application in cartilage regenerative medicine.

Clinical application of irradiated homograft costal cartilage granule transplantation for the pyriform aperture sunken and deformity in the facial contour

Objective To explore the clinical application of irradiated homograft costal cartilage (IHCC) granule transplantation for the pyriform aperture sunken and deformity in the facial contour. Methods From August 2016 to November 2017, a total of 29 female patients were received IHCC transplantation to the face contour. The standardized photos were taken before and after the operation and then survey of patient satisfaction was conducted. Results In a total of 29 patients, the postoperative 21 patients were satisfied with the IHCC transplantation. After the operation, 5 cases were slightly highlighted and obvious bundles could be touched in the deep. At the early stage of operation, the cartilage particles were smoothed by means of manipulation. 3 cases were not satisfied with full effect postoperatively and satisfied after secondary transplantation. Conclusions The IHCC granule transplantation for pyriform aperture sunken and deformity can increase the overall sense of facial coordination and anti-aging, and is a safe and effective treatment. Key words: Costal cartilage; Allografts; Irradiated homograft costal cartilage; Face contour; Pyriform aperturesunken deformity

Chondrocytes From Device-Minced Articular Cartilage Show Potent Outgrowth Into Fibrin and Collagen Hydrogels.

Background:Transplantation of autologous minced cartilage is an established procedure to repair chondral lesions. It relies on the migration of chondrocytes out of cartilage particles into a biomaterial. So far, there is no efficient way to finely mince cartilage. No consensus exists on the nature of the biomaterial to be used to promote chondrocyte migration.Purpose/Hypothesis:This study aimed to investigate the potential clinical use of a custom-made mincing device as well as a possible alternative biomaterial to fibrin glue. The device was tested for its effect on chondrocyte viability and on subsequent chondrocyte migration into either a fibrin or a collagen gel. We hypothesized that device mincing would allow finer cutting and consequently more cell migration and that the gelation mechanism of the collagen biomaterial, which uses the clotting of platelet-rich plasma, would enhance matrix production by outgrown chondrocytes.Study Design:Controlled laboratory study.Methods:Cartilage from 12 patients undergoing knee arthroplasty was taken from the femoral condyles and subsequently either hand minced or device minced. The viability and the degree of outgrowth were quantified with live/dead assay on the generated cartilage particles and on the gels in which these particles were embedded, respectively. Matrix deposition in the biomaterials by the outgrown cells was investigated with histology.Results:The device allowed rapid mincing of the cartilage and produced significantly smaller pieces than hand mincing. The initial chondrocyte viability in cartilage particles dropped by 25% with device mincing as compared with no mincing. However, the viability in hand-minced, device-minced, and unminced samples was no longer different after 7 and 28 days in culture. Outgrowth scores were similar among the 3 groups. Fibrin and collagen biomaterials equally supported chondrocyte outgrowth and survival, but neither promoted matrix deposition after in vitro culture.Conclusion:The outgrowth potential, the viability after 28 days in culture, and the matrix deposition were not different between the mincing techniques and the tested biomaterials, yet device mincing is faster and results in significantly smaller cartilage particles.Clinical Relevance:Device mincing could become the standard method to mince cartilage for second-generation cartilage repair techniques.

Integration Capacity of Human Induced Pluripotent Stem Cell-Derived Cartilage.

New cell and tissue sources are needed for the regenerative treatment of articular cartilage damage. Human induced pluripotent stem cells (hiPSCs) are an abundant cell source due to their self-renewal capacity. Hyaline cartilage tissue particles derived from hiPSCs (hiPS-Carts), 1–3 mm in diameter, are one candidate source that can be used for transplantation. When transplanted to fill the defects of articular cartilage, hiPS-Carts form a repair tissue by integrating with each other and with adjacent host tissue. In this study, we analyzed the integration capacity using an in vitro model and found that hiPS-Carts spontaneously integrate with each other in vitro. hiPS-Carts consist of cartilage at the center and perichondrium-like membrane that wraps around the cartilage. The integration started at the perichondrium-like membrane at around 1 week. Then, the integration progressed to the cartilage within 4–8 weeks. RNA sequencing analysis identified a higher expression of FGF18 in the perichondrium-like membrane in hiPS-Carts compared with the central cartilage. The addition of FGF18 to the model accelerated the integration of hiPS-Carts, whereas the addition of a FGFR inhibitor inhibited it. These results suggest that FGF18 secreted from the perichondrium-like membrane plays a role in the integration of hiPS-Carts. Understanding the integration mechanism of hiPS-Carts is expected to contribute to the realization of regenerative treatment for patients with articular cartilage damage.Impact StatementCartilage particles derived from human induced pluripotent stem cells (hiPS-Carts) are one candidate source for transplants for treatment of articular cartilage damage. This study shows that hiPS-Carts integrate with each other in an in vitro model and analyzed the course of the integration. The integration starts at the perichondrium-like membrane at around 1 week and then progresses to the central cartilage within 4–8 weeks. The results indicate that FGF18 secreted from the perichondrium-like membrane accelerates the initial step of integration. The findings contribute to understanding how hiPS-Carts form repair tissue and provide clue to accelerate healing after transplantation.

Toward understanding the role of cartilage particulates in synovial inflammation

Arthroscopy with lavage and synovectomy can remove tissue debris from the joint space and the synovial lining to provide pain relief to patients with osteoarthritis (OA). Here, we developed an invitro model to study the interaction of cartilage wear particles with fibroblast-like synoviocytes (FLS) to better understand the interplay of cartilage particulates with cytokines on cells of the synovium. In this study sub-10μm cartilage particles or 1μm latex particles were co-cultured with FLS ±10ng/mL interleukin-1α (IL-1α) or tumor necrosis factor-α (TNF-α). Samples were analyzed for DNA, glycosaminoglycan (GAG), and collagen, and media samples were analyzed for media GAG, nitric oxide (NO) and prostaglandin-E2 (PGE2). The nature of the physical interaction between the particles and FLS was determined by microscopy. Both latex and cartilage particles could be phagocytosed by FLS. Cartilage particles were internalized and attached to the surface of both dense monolayers and individual cells. Co-culture of FLS with cartilage particulates resulted in a significant increase in cell sheet DNA and collagen content as well as NO and PGE2 synthesis compared to control and latex treated groups. The proliferative response of FLS to cartilage wear particles resulted in an overall increase in extracellular matrix (ECM) content, analogous to the thickening of the synovial lining observed in OA patients. Understanding how cartilage particles interface with the synovium may provide insight into how this interaction contributes to OA progression and may guide the role of lavage and synovectomy for degenerative disease.

Promoting chondroinduction with naturally derived hydrogel pastes

Event Abstract Back to Event Promoting chondroinduction with naturally derived hydrogel pastes Emily Beck1, Marilyn Barragan2, Madeleine H. Tadros3, Stevin H. Gehrke1, 4, Sarah L. Kieweg1, 5, Cory J. Berkland1, 4, 6 and Michael S. Detamore1, 4 1 University of Kansas, Bioengineering Program, United States 2 University of Kansas, Department of Molecular Biosciences, United States 3 Rice University, Department of Chemical and Biomolecular Engineering, United States 4 University of Kansas, Department of Chemical and Petroleum Engineering, United States 5 University of Kansas, Department of Mechanical Engineering, United States 6 University of Kansas, Department of Pharmaceutical Chemistry, United States Introduction: ECM-based materials are gaining wide attention in the tissue engineering field due to their ability to support cellular attachment, growth, and differentiation[1]. Although ECM materials have been incorporated into hydrogels and show great promise, hydrogels also have one major drawback because their precursors are liquid solutions that are prone to leaking after surgical placement[2]. Therefore, in this work, we mixed porcine devitalized cartilage (DVC) particles into methacrylated, solubilized, devitalized, porcine cartilage (MeSDVC) to achieve a hydrogel precursor that was not only paste-like to overcome the drawbacks of traditional hydrogels, but additionally to achieve a hydrogel that would induce chondrogensis and could be further crosslinked and analyzed. Methods: Porcine cartilage that was devitalized and cryoground into a fine powder was mixed with devitalized cartilage that was solubilized and then methacrylated, and the precursors were analyzed rheologically for yield stress. Formulations tested rheologically were 5% DVC, 10% DVC, 10% MeSDVC, 10% MeSDVC 10% DVC, and 10% MeSDVC 10% DVC + TGF-β3. The formulations containing MeSDVC were then mixed with rat bone marrow stem cells and UV crosslinked to further characterize them as solids. The gels were cultured in vitro for 6 weeks, where chondrogenic gene expression, the compressive modulus, swelling, and histology were also analyzed. Results and Discussion: All formulations tested exhibited a yield stress, where the yield stress of 10% MeSDVC and 10% DVC were 725 ± 55 and 57.9 ± 0.3, respectively. When 10% MeSDVC and 10% DVC were mixed, the yield stress increased by a factor of 2.6 compared to 10% MeSDVC alone (p<0.05). Because the yield stress is not additive, it suggests that there is some physiological or chemical interaction between MeSDVC and DVC that is creating the synergistic yield stress effect. After crosslinking, the only gels that were noted to not have contraction throughout culture were the gels containing DVC particles. At the 2 week and 3 week time points, Sox-9 and aggrecan expression were significantly higher in the DVC groups (p<0.05) and by 6 weeks, the relative collagen II expressions of the 10% MeSDVC 10% DVC and the 10% MeSDVC 10% DVC + TGF-β3 groups were 78 and 40 fold higher than 10% MeSDVC alone, respectively (p<0.05). The compressive moduli of all but one group ranged between 70 and 160 kPa, although the 20% MeSDVC acellular group had a modulus of 680 ± 130 kPa, which is much closer to that of native cartilage. Finally, aggrecan immunohistological staining showed increased staining around the cells at 6 weeks in the growth factor supplemented and the 10% MeSDVC group. Conclusion: Given that the mixtures of MeSDVC and DVC combined yielded hydrogel precursors that exhibited a yield stress and crosslinked to form hydrogels that did not contract over time, stimulated chondroinductive gene expression, as well as had a compressive moduli on the same order of magnitude as native cartilage, these MeSDVC and DVC mixtures may be promising materials for translational cartilage tissue engineering applications. NIH S10 RR024664; NSF Graduate Research Fellowship (E.B.); NSF Major Research Instrumentation Grant (0320648); Kansas Bioscience Authority Rising Star Award (M.D.); NIH R01 DE022472 (C.B.)

Decellularized cartilage may be a chondroinductive material for osteochondral tissue engineering.

Extracellular matrix (ECM)-based materials are attractive for regenerative medicine in their ability to potentially aid in stem cell recruitment, infiltration, and differentiation without added biological factors. In musculoskeletal tissue engineering, demineralized bone matrix is widely used, but recently cartilage matrix has been attracting attention as a potentially chondroinductive material. The aim of this study was thus to establish a chemical decellularization method for use with articular cartilage to quantify removal of cells and analyze the cartilage biochemical content at various stages during the decellularization process, which included a physically devitalization step. To study the cellular response to the cartilage matrix, rat bone marrow-derived mesenchymal stem cells (rBMSCs) were cultured in cell pellets containing cells only (control), chondrogenic differentiation medium (TGF-β), chemically decellularized cartilage particles (DCC), or physically devitalized cartilage particles (DVC). The chemical decellularization process removed the vast majority of DNA and about half of the glycosaminoglycans (GAG) within the matrix, but had no significant effect on the amount of hydroxyproline. Most notably, the DCC group significantly outperformed TGF-β in chondroinduction of rBMSCs, with collagen II gene expression an order of magnitude or more higher. While DVC did not exhibit a chondrogenic response to the extent that DCC did, DVC had a greater down regulation of collagen I, collagen X and Runx2. A new protocol has been introduced for cartilage devitalization and decellularization in the current study, with evidence of chondroinductivity. Such bioactivity along with providing the ‘raw material’ building blocks of regenerating cartilage may suggest a promising role for DCC in biomaterials that rely on recruiting endogenous cell recruitment and differentiation for cartilage regeneration.

Study on the feasibility of tissue engineering cartilage constructed by cartilage particle and bionic sol matrix composite

Objective To explore the feasibility of constructing tissue engineered cartilage by using the cartilage particle as seed cells and collagenⅡ- hyaluronic acid- chondroitin sulfate(COLⅡ- HA- CS) bionic matrix sol. Methods Cartilage particles were acquired from 5 months old Guizhou minipig knee joint; particles with different diameters were acquired by sieve filtration.The proliferation and adhesion of chondrocytes were evaluated at day 1,3,and 7.The bionic sol matrix at final concentration of 10 g/L was prepared by dissolving COLⅡ- HA- CS(mass ratio:8.0∶1.5∶0.5)in 0.15 mol/L HCl; The quality of prepared gel was examined.The cartilage particles with the diameter of strongest proliferation rate was seeded into COLⅡ/ HA/CSsol mix at 5×107/L the in vitro results were evaluated by Living cell immunofluorescence and histological observation after 1 week. Results Cartilage particles with different sizes all developed visible chondrocytes adherence and proliferation, and particle size less than 120μm showed strongest chondrocytes proliferation ability, the difference is statistically significant(P< 0.05). Neutralized COLⅡ/HA/CS bionic sol matrix showed good flowability at low temperature;and transferred to gel at 37 ℃for 6 min.After 1 week the cultured cartilage particles/gel complex showed transparent chondroid appearance, smooth surface and certain level of flexibility; Immunofluorescence observation showed good chondrocytes activity; Histological observation demonstrated chondrocytes were evenly distributed with good viability. Conclusion It reached the best proliferation of chondrocytes when particle size was less than 120μm; Cartilage particles and COLⅡ/ HA/CSbionic matrix sol are feasible to culture and construct bionic cartilage in vitro. Key words: Cartilage particles; Bionic matrix sol; Tissue engineering cartilage

Long-Term Expandable SOX9+ Chondrogenic Ectomesenchymal Cells from Human Pluripotent Stem Cells

SummaryHere we report the successful generation and long-term expansion of SOX9-expressing CD271+PDGFRα+CD73+ chondrogenic ectomesenchymal cells from the PAX3/SOX10/FOXD3-expressing MIXL1−CD271hiPDGFRαloCD73− neural crest-like progeny of human pluripotent stem cells in a chemically defined medium supplemented with Nodal/Activin/transforming growth factorβ (TGFβ) inhibitor and fibroblast growth factor (FGF). When “primed” with TGFβ, such cells efficiently formed translucent cartilage particles, which were completely mineralized in 12 weeks in immunocompromized mice. The ectomesenchymal cells were expandable without loss of chondrogenic potential for at least 16 passages. They maintained normal karyotype for at least 10 passages and expressed genes representing embryonic progenitors (SOX4/12, LIN28A/B), cranial mesenchyme (ALX1/3/4), and chondroprogenitors (SOX9, COL2A1) of neural crest origin (SOX8/9, NGFR, NES). Ectomesenchyme is a source of many craniofacial bone and cartilage structures. The method we describe for obtaining a large quantity of human ectomesenchymal cells will help to model craniofacial disorders in vitro and potentially provide cells for the repair of craniofacial damage.

Iodoacetate and allogenous cartilage particles as models for arthritis induction in equine

Experimental models of osteoarthritis (OA) have been widely developed in different animal species, because of the high incidence of osteoarthritis diseases in humans and animals. To date, no ideal OA animal model has been reported. The present study compare different osteoarthritis models to determine which one is suitable for inducing experimental equine OA. Fifteen donkeys were divided into three equal groups (n=5). The radio carpal joints of the right forelimb of 15 donkeys were injected with 25mg monoiodoacetate (MIA) (group A), 50mg allogenous cartilage particles (ACP) (group B), or vehicle solution (group C) over a period of 70days. Osteoarthritis induction was evaluated weekly through lameness score, carpal circumference, joint flexion angel, synovial fluid analysis (total protein and WBC count), and radiology. Animal were euthanized and joints histopathology were performed at 70days. Lameness score and joint circumference was increased in both group A and B however joint flexion angel was decreased compared to group C (p<0.05). Osteophytes were observed in MIA injected joints only accompanied with subchondral bone sclerosis. Cartilage damage was observed grossly and histologically in Group A together with synovial membrane fibrosis. Group B had on cartilage damage grossly however histological examination revealed some cartilage surface discontinuity with synovial membrane edema. Injection of monoiodoacetate in the donkey is a successful model to create the acute clinical signs of joint disease as well as cartilage damage. However, allogenous cartilage particles injection need more investigation to be applied.

Small molecule-directed specification of sclerotome-like chondroprogenitors and induction of a somitic chondrogenesis program from embryonic stem cells.

Pluripotent embryonic stem cells (ESCs) generate rostral paraxial mesoderm-like progeny in 5-6 days of differentiation induced by Wnt3a and Noggin (Nog). We report that canonical Wnt signaling introduced either by forced expression of activated β-catenin, or the small-molecule inhibitor of Gsk3, CHIR99021, satisfied the need for Wnt3a signaling, and that the small-molecule inhibitor of BMP type I receptors, LDN193189, was able to replace Nog. Mesodermal progeny generated using such small molecules were chondrogenic in vitro, and expressed trunk paraxial mesoderm markers such as Tcf15 and Meox1, and somite markers such as Uncx, but failed to express sclerotome markers such as Pax1. Induction of the osteochondrogenically committed sclerotome from somite requires sonic hedgehog and Nog. Consistently, Pax1 and Bapx1 expression was induced when the isolated paraxial mesodermal progeny were treated with SAG1 (a hedgehog receptor agonist) and LDN193189, then Sox9 expression was induced, leading to cartilaginous nodules and particles in the presence of BMP, indicative of chondrogenesis via sclerotome specification. By contrast, treatment with TGFβ also supported chondrogenesis and stimulated Sox9 expression, but failed to induce the expression of Pax1 and Bapx1. On ectopic transplantation to immunocompromised mice, the cartilage particles developed under either condition became similarly mineralized and formed pieces of bone with marrow. Thus, the use of small molecules led to the effective generation from ESCs of paraxial mesodermal progeny, and to their further differentiation in vitro through sclerotome specification into growth plate-like chondrocytes, a mechanism resembling in vivo somitic chondrogenesis that is not recapitulated with TGFβ.

Carboxyl-modified single-wall carbon nanotubes improve bone tissue formation in vitro and repair in an in vivo rat model.

The clinical management of bone defects caused by trauma or nonunion fractures remains a challenge in orthopedic practice due to the poor integration and biocompatibility properties of the scaffold or implant material. In the current work, the osteogenic properties of carboxyl-modified single-walled carbon nanotubes (COOH–SWCNTs) were investigated in vivo and in vitro. When human preosteoblasts and murine embryonic stem cells were cultured on coverslips sprayed with COOH–SWCNTs, accelerated osteogenic differentiation was manifested by increased expression of classical bone marker genes and an increase in the secretion of osteocalcin, in addition to prior mineralization of the extracellular matrix. These results predicated COOH–SWCNTs’ use to further promote osteogenic differentiation in vivo. In contrast, both cell lines had difficulties adhering to multi-walled carbon nanotube-based scaffolds, as shown by scanning electron microscopy. While a suspension of SWCNTs caused cytotoxicity in both cell lines at levels >20 μg/mL, these levels were never achieved by release from sprayed SWCNTs, warranting the approach taken. In vivo, human allografts formed by the combination of demineralized bone matrix or cartilage particles with SWCNTs were implanted into nude rats, and ectopic bone formation was analyzed. Histological analysis of both types of implants showed high permeability and pore connectivity of the carbon nanotube-soaked implants. Numerous vascularization channels appeared in the formed tissue, additional progenitor cells were recruited, and areas of de novo ossification were found 4 weeks post-implantation. Induction of the expression of bone-related genes and the presence of secreted osteopontin protein were also confirmed by quantitative polymerase chain reaction analysis and immunofluorescence, respectively. In summary, these results are in line with prior contributions that highlight the suitability of SWCNTs as scaffolds with high bone-inducing capabilities both in vitro and in vivo, confirming them as alternatives to current bone-repair therapies.