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3D composite engineered using supercritical CO2 decellularized porcine cartilage scaffold

3D composite engineered using supercritical CO2 decellularized porcine cartilage scaffold, chondrocytes, and PRP: Role in articular cartilage regeneration

At present, no definitive treatment for articular cartilage defects has been perfected. Most of the previous treatments involved multiple drilling and microfracture over defect sites with repair‐related substances, which poses a limited therapeutic effect. End‐stage therapy includes artificial knee joint replacement. In this study, they prepared a novel decellularized natural cartilage scaffold from porcine articular cartilage by supercritical CO2 extraction technology and three‐dimensional (3D) composites made using decellularized porcine cartilage graft (dPCG) as scaffolds, platelet‐rich plasma (PRP), thrombin as signals and chondrocytes as cells for the treatment of articular cartilage defects. In this study, in vitro and in vivo cartilage regeneration and the expression of chondrogenic markers were examined. Decellularized cartilage graft (dPCG) was evaluated for the extent of cell and DNA removal. Residual cartilage ECM structure was confirmed to be type II collagen by SDS PAGE and immunostaining. The new 3D composite with dPCG (100 mg and 2 × 106 chondrocytes) scaffold promotes chondrogenic marker expression in vitro. They found that the in vivo 3D composite implanted cartilage defect showed significant regeneration relative to the blank and control implant. Immunohistochemical staining showed increase of expression including Collagen type II and aggrecan in 3D composite both in vitro and in vivo studies. In this study, the bioengineered 3D composite by combining dPCG scaffold, chondrocytes, and PRP facilitated the chondrogenic marker expression in both in vitro and in vivo models with accelerated cartilage regeneration. This might serve the purpose of clinical treatment of large focal articular cartilage defects in humans in the near future.


Cartilage tissue engineering (CTE) aims to produce cartilage‐like tissue substitutes by merging the suitable cells, scaffolds, and bioactive molecules to promote repairing articular cartilage injury. Autologous chondrocytes are the best choice of cells for cartilage repair due to their inherent properties related to cell function and immune compatibility. However, there are limited chondrocytes accessibility from patients, and if available, it also requires extensive expansion into a two‐dimensional (2D) monolayer culture of chondrocytes.




Characterization of dPCG by (a) H&E staining of normal cartilage and dPCG. (b) DAPI staining of normal cartilage and dPCG. (c) Alcian blue staining of normal cartilage and dPCG. (d) Alpha‐galactosidase immunostaining of normal cartilage and dPCG. (e) Quantification of DNA in normal cartilage and dPCG. (f) Quantification of GAGs in normal cartilage and dPCG.


Chondrocytes exhibit an extensive loss of the original tissue‐specific phenotype during the expansion of chondrocytes, leading to fibroblast‐like phenotype. At present three‐dimensional (3D) culture platforms are employed to restore chondrogenic phenotype, which mimics the cellular microenvironment similar to that in vivo. The choice of biomaterials used for CTE varied in composition, structure, biodegradability, and biomechanical properties. Natural scaffolds are commonly used biomaterial such as collagen type I, laminin, and gelatin, which provide chemical signals, principally ECM binding motifs.



Chondrocytes‐3D composite (encapsulated with 2 × 106 porcine chondrocytes) immunostaining of anabolic factors, type II collagen (a), aggrecan (b), semi quantification of type II collagen (c), aggrecan (d).

Acellular cartilage matrix derived from the cartilage would make ideal scaffolds for tissue engineering. Numerous decellularization methods are used in cartilage scaffold preparation including physical, chemical, and enzymatic treatments, however, these processes involve certain disadvantages such as traces of impurities and loss of scaffold structure. These disadvantages can be overcome by using supercritical carbon dioxide (SCCO2) extraction technology. SCCO2 employs mild critical coordinates, such as pressure at 7.38 MPa and temperature at 31°C, which can be easily accomplished and well‐suited for biological material preparation. In addition, the SCCO2 process leaves no chemical solvent residue, solubilizes and removes oil and lipids, and hence has the advantages of both bactericidal and viral inactivation effects, which makes the processed biomaterials better in biocompatibility.




3D composite implantation to full‐thickness osteochondral defect. Representative safranin‐O staining photomicrograph. (a) Group 1—PT (autologous PRP and thrombin gel). (b) Group 2—PTCh (autologous PRP, thrombin gel, and chondrocytes). (c) Group 3—PTC (autologous PRP, thrombin gel, and dPCG scaffold). (d) Group 4—PTChC (autologous PRP, thrombin gel, chondrocytes, and dPCG scaffold). (e) ICRS gross morphology score. (f) ICRS histological score.


Advantages of SCCO2 technique include that it is natural, safe, nontoxic, noncorrosive, nonflammable, easily accessible, cost‐effective, destroys, and eliminates pathogens . There are no known disadvantages of SCCO2 technique concerning the manufacture of decellularized collagen materials, including cartilage in literature.



3D composite implantation to full‐thickness osteochondral defect. Expression of type II collagen (a–d). Semi quantification of type II collagen (e).


In the present study, they decellularized the porcine articular cartilage from femoral condyle using the SCCO2 extraction technique and examined the complete decellularization, chemical nature, and scaffold structure of the cartilage scaffold. In addition, they engineered 3D composites using SCCO2 decellularized porcine cartilage graft (dPCG), platelet‐rich plasma (PRP), thrombin, and porcine articular chondrocytes and evaluated the in vitro efficacy of the 3D composites for the proliferation of chondrocytes and the expression of collagen type II and aggrecan. Furthermore, the regenerative role of the 3D composite was evaluated in porcine full‐thickness osteochondral defect model. The current study aimed to demonstrate the vital role of dPCG scaffold in the chondrogenesis process in a 3D construct that mimics the microenvironments of cartilage tissue1.


3D composite implantation to full‐thickness osteochondral defect.

3D composite implantation to full‐thickness osteochondral defect. Representative safranin‐O staining photomicrograph. (a) Group I—dPCG (autologous PRP, thrombin gel, and chondrocytes). (b) Group II—collagen matrix (autologous PRP, thrombin gel, and chondrocytes). (c) ICRS gross morphology score. (d) ICRS histological score.


  1. Chen, Y.‐T., Lee, H.‐S., Hsieh, D.‐J., Periasamy, S., Yeh, Y.‐C., Lai, Y.‐P. and Tarng, Y.‐W. (2021), 3D composite engineered using supercritical CO2 decellularized porcine cartilage scaffold, chondrocytes, and PRP: Role in articular cartilage regeneration. J Tissue Eng Regen Med, 15: 163-175. https://doi.org/10.1002/term.3162