A series of novel decellularized porcine collagen bone graft (DPB) materials in a variety of shapes and sizes were developed by the supercritical carbon dioxide (SCCO2) extraction technique. The complete decellularization of DPB was confirmed by hematoxylin and eosin staining, 4,6‐diamidino‐2‐phenylindole (DAPI) staining, and residual DNA analysis. The native intact collagen remained in the DPB after the SCCO2 process was confirmed by Masson trichrome staining. The physicochemical characteristics of DPB were investigated by scanning electron microscopy and x‐ray diffraction. The cytotoxicity and biocompatibility tests according to ISO10993 and its efficacy for bone regeneration in osteochondral defects in rabbits were evaluated. The rabbit pyrogen test confirmed DPB was non‐toxic. In vitro and in vivo biocompatibility tests of the DPB did not show any toxic or mutagenic effects. The bone regeneration potential of the DPB presented no significant histological differences compared to commercially available deproteinized bovine bone. In conclusion, DPB produced by SCCO2 exhibited similar chemical characteristics to human bone, no toxicity, good biocompatibility, and enhanced bone regeneration in rabbits comparable to that of deproteinized bovine bone. Results from this study could shed light on the potential application of the SCCO2 extraction technique to generate a native decellularized scaffold for bone tissue regeneration in human clinical trials.

Extracellular matrix (ECM) scaffolds are extensively used in tissue engineering studies and numerous clinical applications for tissue and organ reconstructions. Due to the global severe shortage of human tissues and organs, xenogeneic biomaterials are a common source for human tissue engineering and regenerative medicine applications. Traditional methods for decellularization often disrupt the 3D architecture and damage the structural integrity of the ECM scaffold. To efficiently obtain natural ECM scaffolds from animal tissues and organs with intact architecture, we have developed a platform decellularization process using supercritical CO2 and tested its potential application in tissue engineering. A combination of human mesenchymal stem cells with a decellularized dermal matrix scaffold allowed complete regeneration of skin structure in a porcine full-thickness wound model.

Orbital floor fractures subsequently lead to consequences such as diplopia and enophthalmos. The graft materials used in orbital floor fractures varied from autografts to alloplastic grafts, which possess certain limitations. In the present study, a novel porcine bone matrix decellularized by supercritical CO2 (scCO2), ABCcolla® Collagen Bone Graft, was used for the reconstruction of the orbital framework. The study was approved by the institutional review board (IRB) of Kaohsiung Medical University Chung-Ho Memorial Hospital (KMUH). Ten cases underwent orbital floor reconstruction in KMUH in 2019. The orbital defects were fixed by the implantation of the ABCcolla® Collagen Bone Graft. Nine out of ten cases used 1 piece of customized ABCcolla® Collagen Bone Graft in each defect. The other case used 2 pieces of customized ABCcolla® Collagen Bone Graft in one defect area due to the curved outline of the defect. In the outpatient clinic, all 10 cases showed improvement of enophthalmos on CT (computerized tomography) at week 8 follow-up. No replacement of implants was needed during follow-ups. To conclude, ABCcolla® Collagen Bone Graft proved to be safe and effective in the reconstruction of the orbital floor with high accessibility, high stability, good biocompatibility, low infection rate and low complication rate.

Damage to abdominal wall integrity occurs in accidents, infection and herniation. Repairing the hernia remains to be one of the most recurrent common surgical techniques. Supercritical carbon dioxide (SCCO2) was used to decellularize porcine skin to manufacture acellular dermal matrix (ADM) for the reparation of full-thickness abdominal wall defects and hernia. The ADM produced by SCCO2 is chemically equivalent and biocompatible with human skin. The ADM was characterized by hematoxylin and eosin (H&E) staining, 4,6-Diamidino-2-phenylindole, dihydrochloride (DAPI) staining, residual deoxyribonucleic acid (DNA) contents and alpha-galactosidase (α-gal staining), to ensure the complete decellularization of ADM. The ADM mechanical strength was tested following the repair of full-thickness abdominal wall defects (4 × 4 cm) created on the left and right sides in the anterior abdominal wall of New Zealand White rabbits. The ADM produced by SCCO2 technology revealed complete decellularization, as characterized by H&E, DAPI staining, DNA contents (average of 26.92 ng/mg) and α-gal staining. In addition, ADM exhibited excellent performance in the repair of full-thickness abdominal wall defects. Furthermore, the mechanical strength of the reconstructed abdominal wall after using ADM was significantly (p < 0.05) increased in suture retention strength (30.42 ± 1.23 N), tear strength (63.45 ± 7.64 N and 37.34 ± 11.72 N) and burst strength (153.92 ± 20.39 N) as compared to the suture retention (13.33 ± 5.05 N), tear strength (6.83 ± 0.40 N and 15.27 ± 3.46 N) and burst strength (71.77 ± 18.09 N) when the predicate device materials were concomitantly tested. However, the efficacy in hernia reconstruction of ADM is substantially equivalent to that of predicate material in both macroscopic and microscopic observations. To conclude, ADM manufactured by SCCO2 technology revealed good biocompatibility and excellent mechanical strength in post-repair of full-thickness abdominal wall defects in the rabbit hernia model.

About 30–50% of oral cancer patients require mandibulectomy and autologous fibula reconstruction. Autograft is the gold standard choice because of its histocompatibility; however, it requires additional surgery from the patient and with possible complications such as loss of fibula leading to calf weakening in the future. Allograft and xenograft are alternatives but are susceptible to immune response. Currently, no personalized bone xenografts are available in the market for large fascial bone defects. In addition, a large-sized complex shape bone graft cannot be produced directly from the raw material. We propose the use of porcine bones with 3D CAD/CAM carving to reconstruct a personalized, wide range and complex-shaped bone. We anticipate that patients can restore their native facial appearance after reconstruction surgery. Supercritical CO2 (SCCO2) technology was employed to remove the cells, fat and non-collagenous materials while maintaining a native collagen scaffold as a biomedical device for bone defects. We successfully developed 3D CAD/CAM carved bone matrices, followed by SCCO2 decellularization of those large-sized bones. A lock-and-key puzzle design was employed to fulfil a wide range of large and complex-shaped maxillofacial defects. To conclude, the 3D CAD/CAM carved bone matrices with lock and key puzzle Lego design were completely decellularized by SCCO2 extraction technology with intact natural collagen scaffold. In addition, the processed bone matrices were tested to show excellent cytocompatibility and mechanical stiffness. Thus, we can overcome the limitation of large size and complex shapes of xenograft availability. In addition, the 3D CAD/CAM carving process can provide personalized tailor-designed decellularized bone grafts for the native appearance for maxillofacial reconstruction surgery for oral cancer patients and trauma patients.