Recellularization of Acellular Xeno Kidney Scaffold: An In Vivo Method to Generate Bioartificial Kidney.

A significant hurdle for kidney tissue engineering is reproducing the complex three-dimensional structure of the kidney. In our study, a stepwise approach of generating a reproducible Xeno kidney scaffold from a goat kidney is described, which can be implanted and recellularized by host cells. We have proposed a combination of sodium dodecyl sulfate and Triton-X-100-based protocol to generate a reproducible Xeno kidney scaffold, which was then analyzed by histology, DNA quantification, SEM, and renal angiography. Further, a small portion from the cortico-medullar region of the acellular scaffold was implanted in the rat's kidney subcapsular pocket for a period of 1 month, to check the recruitment of host cells into the scaffold. Post implantation, the extracellular matrix of the scaffold was well preserved and it did not induce any damage or inflammation in the native kidney. Implantation of the Xeno scaffold resulted in apparent early vascularization which helped in the recruitment of the host cells, which was characterized by histology, immunohistochemistry, and scanning electron microscopy. Implanted Xeno scaffold showed AQP-1, Nephrin, α-SMA, and VEGF expression in proximal tubules and renal glomerulus. Importantly, Ki-67 and WTAP-expressing cells were also observed near proximal tubules suggesting a high level of proliferation in the scaffold. Thus, showing the potential of Xeno kidney development that can be recellularized by the host cell to engineer into a functional kidney.

Heparin coated decellularized xenogeneic small diameter vascular conduit for vascular repair with early luminal reendothelialization.

Small diameter vascular graft is a clinical need in cardiovascular disease (CAD) and peripheral atherosclerotic diseases (PAD). Autologous graft has limitations in availability and harvesting surgery. To make luminal surface modification with heparin coating in xenogeneic small diameter vascular graft. We constructed a conduit from decellularized human saphenous vein (HSV) matrices in small diameter vascular graft (< 0.8 mm diameter). Luminal surface modification was done with heparin coating for transplantation in the rat femoral artery. Biocompatibility of conduit was checked in Chorioallantoic Membrane (CAM) assay and in vivo. The blood flow rate in conduit grafts was measured, and immuno-histological analysis was performed. CAM assay and in vivo biocompatibility test showed cellular recruitment in the HSV scaffold. Heparin binding was achieved on the luminal surface. After three months of transplantation surgery neo-intimal layer was formed in the graft. The graft was patent for two weeks after surgery. There were no statistically differences between blood flow rate in graft (at proximal end 0.5 ± 0.01 m/s and at distal end 0.4 ± 0.01 m/s (n = 6)) and native artery (0.6 ± 0.1 m/second, (n = 3)). Biomarkers of endothelial cells, medial smooth muscle cells, and angiogenesis were observed in the transplanted graft. Our study demonstrates that xenogeneic decellularized vascular grafts with surface modification with heparin coating could be useful for the replacement of small diameter vessels.

Tissue engineering of human ear pinna.

Auricular deformities (Microtia) can cause physical, social as well as psychological impacts on a patient's wellbeing. Biofabrication of a complex structure such as ear pinna is not precise with currently available techniques. These limitations can be overcome with the help of tissue engineering. In this article, the authors presented molding and three dimensional (3D) printing to generate a flexible, human size ear pinna. The decellularization of goat ear cartilage protocol and bioink alkaline digestion protocol was followed to yield complete removal of all cellular components without changing the properties of the Extra Cellular Matrix (ECM). Decellularized scaffold used in molding technology and 3D printing technology Computer-Aided Design /Stereolithography (CAD/STL) uses bioink to construct the patient-specific ear. In vivo biocompatibility of the both ear pinnae showed demonstrable recellularization. Histology and scanning electron microscopy analysis revealed the recellularization of cartilage-specific cells and the development of ECM in molded and 3D printed ear pinna after transplantation. Both the techniques provided ideal results for mechanical properties such as elasticity. Vascular Associated Protein expression revealed specific vasculogenic pattern (angiogenesis) in transplanted molded pinna. Chondrocyte specific progenitor cells express CD90+ which highlighted newly developed chondrocytes in both the grafts which indicated that the xenograft was accepted by the rat. Transplantation of molded as well as 3D ear pinna was successful in an animal model and can be available for clinical treatments as a medical object to cure auricular deformities.

Tissue engineered human ear pinna derived from decellularized goat ear cartilage: clinically useful and biocompatible auricle construct.

Surgery of the entire ear pinna even today presents a challenge to reconstructive surgeons, in the absence of a universally acceptable, quality construct for clinical use. In this article, the authors present a technique to generate a flexible, human size ear with the aim to meet this limitation for ear reconstructive surgeries. The construct was engineered by using a decellularized goat ear cartilage. This was characterized by hematoxylin-eosin (H/E), diamidino-2-phenylindole (DAPI), Masson's trichrome (MT), Alcian Blue (AB) staining and Scanning Electron Microscopy (SEM) for extracellular matrix (ECM) analysis. The decellularization protocol followed yielded complete removal of all cellular components without changing the properties of the ECM. In vivo biocompatibility of the ear pinna showed demonstrable recellularization. Recellularization was tracked using HE, DAPI, MT, AB staining, toluidine staining, SEM, vascular-associated protein (VAP) and CD90+ expressing cells. VAP expression revealed specific vasculogenic pattern (angiogenesis). CD90+ expression reflected the presence of the stromal cell. The graft maintained the properties of ECM and displayed chondrocyte recruitment. In summary, the decellularized goat ear pinna (cartilage) exhibited xenograft biocompatibility, stable mechanical properties and in vivo chondrocyte recruitment. Subsequently developed tissue-engineered ear pinna offer potential for cartilage flexibility and individualization of ear shape and size for clinical application.

A biologically functional bioink based on extracellular matrix derived collagen for 3D printing of skin.

Over the past few years, several advancements have been made to develop artificial skin that mimics human skin. Artificial skin manufactured using 3D printing technology that includes all epidermal and dermal components, such as collagen, may offer a viable solution. The skin-specific bioink was derived from digested chicken skin and incorporated into PVA (polyvinyl alcohol) and gelatin. The prepared bioink was further analyzed for its structure, stability, biocompatibility, and wound healing potential in in vitro, in ovo, and in vivo models. The 3D-printed skin showed excellent mechanical properties. In vitro scratch assays showed the proliferation and migration of cells within 24 h. In an in ovo assay, the 3D-printed skin facilitated the attachment of cells to the scaffolds. In the animal study, the quick cellular recruitment at the injury site accelerated wound healing. Further, hydroxyproline content was estimated to be 0.9-1.2 mg/ml, and collagen content was 7.5 %, which confirmed the epithelization. The relative expressions of MMP-9, COMP, TNF-α, and IL-6 genes were found to be increased compared to the control. These results demonstrate that 3D bioprinting represents a suitable technology to generate bioengineered skin for therapeutic and industrial applications in an automated manner.

Decellularized small intestine scaffolds: a potential xenograft for restoration of intestinal perforation.

Small intestine perforation is a serious medical condition that requires immediate medical attention. The traditional course of treatment entails resection followed by anastomosis; however, it has complications such as small bowel syndrome (SBS), anastomotic leakage, and fistula formation. Here, a novel strategy is demonstrated, that utilizes the xenogeneic, decellularized goat small intestine as a patch for small intestine regeneration in cases of intestinal perforation. The goat small intestine scaffold underwent sodium dodecyl sulfate decellularization, which revealed consistent, quick, and effective decellularization. Decellularization contributed the least amount of extracellular matrix degradation while maintaining the intestinal architecture. By implanting the decellularized goat small intestine scaffolds (DGSIS) on the chorioallantoic membrane (CAM), no discernible loss of angiogenesis was seen in the CAM region, and this enabled the DGSIS to be evaluated for biocompatibility in ovo. The DGSIS was then xeno-transplanted as a patch on a small intestine perforation rat model. After 30 days post transplant, barium salt used as contrast gastrointestinal X-ray imaging revealed no leakage or obstruction in the small intestine. Histology, scanning electron microscopy, and immunohistochemistry assisted in analyzing the engraftment of host cells into the xeno patch. The xeno-patch expressed high levels of E-cadherin, α-smooth muscle actin (α-SMA), Occludin, Zonnula occluden (ZO-1), Ki 67, and Na+/K+-ATPase. The xeno-patch was consequently recellularized and incorporated into the host without causing an inflammatory reaction. As an outcome, decellularized goat small intestine was employed as a xenograft and could be suitable for regeneration of the perforated small intestine.

Heparin Immobilization of Tissue Engineered Xenogeneic Small Diameter Arterial Scaffold Improve Endothelialization.

BACKGROUND: Autologous vessels graft (Inner diameter < 6 mm) harvesting always challenged during bypass grafting surgery and its complication shows poor outcome. Tissue engineered vascular graft allow to generate biological graft without any immunogenic complication. The approach presented in this study is to induce graft remodeling through heparin coating in luminal surface of small diameter (Inner diameter < 1 mm) decellularized arterial graft. METHODS: Decellularization of graft was done using SDS, combination of 0.5% sodium dodecyl sulfate and 0.5% sodium deoxycholate and only sodium deoxycholate. Decellularization was confirmed on basis of histology, and DAPI. Characterization of extracellular matrix was analyzed using histology and scanning electron microscopy. Surface modification of decellularized vascular graft was done with heparin coating. Heparin immobilization was evaluated by toluidine blue stain. Heparin-coated graft was transplanted end to end anastomosis in femoral artery in rat. RESULTS: Combination of 0.5% sodium dodecyl sulfate and 0.5% Sodium deoxycholate showed complete removal of xenogeneic cells. The heparin coating on luminal surface showed anti-thrombogenicity and endothelialization. Mechanical testing revealed no significant differences in strain characteristics and modulus between native tissues, decellularized scaffolds and transplanted scaffold. Collectively, this study proposed a heparin-immobilized ECM coating to surface modification offering functionalize biomaterials for developing small-diameter vascular grafts. CONCLUSION: We conclude that xenogeneic decellularized arterial scaffold with heparin surface modification can be fabricated and successfully transplanted small diameter (inner diameter < 1 mm) decellularized arterial graft.

3D printing of human ear pinna using cartilage specific ink.

Biofabrication of a complex structure such as ear pinna is not precise with currently available techniques. Auricular deformities (e.g. microtia) can cause physical, social as well as psychological impacts on a patient's wellbeing. Currently available surgical techniques and transplantation methods have many limitations that can be overcome with the help of 3D bioprinting technology. Printable bioink enriched with cartilage-specific extracellular matrix (ECM) synthesis was done by digesting goat ear pinna cartilage and polymerized by adding polyvinyl alcohol and gelatine. Rheological analysis and Fourier-transform infrared spectroscopy were used for the characterization of bioink to get desired viscosity and polymerization. Human ear pinna was printed using extrusion method and computer-aided design, stereolithography software which facilitated the automated printing in relatively less time without continuous monitoring. Thermal degradation of pinna was checked by thermal gravimetric analysis. Biodegradability and swelling of ear pinna were observed for understanding the nature of pinna and the impact of external factors. Reconstructed pinna's biocompatibility was proved byin ovoandin vivostudies. The occurrence of angiogenesis in the grafted ear manifested the capacity of proliferation and engraftment of cartilage cells. Histology and SEM analysis revealed the recellularization and the synthesis of ECM components such as glycosaminoglycan and collagen in transplanted 3D printed ear pinna. The expression of CD90+ which indicated newly synthesized cartilage in the transplanted 3D printed ear pinna. The absence expression of CD14+ also indicated acceptance of xenogenic transplanted 3D printed ear pinna. Transplantation of 3D ear pinna was successful in an animal model and can be utilized as tissue engineered ear bank.