Microstructured hydrogel scaffolds containing differential density interfaces promote rapid cellular invasion and vascularization.

INTRODUCTION: Insufficient vascularization of currently available clinical biomaterials has limited their application to optimal wound beds. We designed a hydrogel scaffold with a unique internal microstructure of differential collagen densities to induce cellular invasion and neovascularization. METHODS: Microsphere scaffolds (MSS) were fabricated by encasing 1% (w/v) type 1 collagen microspheres 50-150 µm in diameter in 0.3% collagen bulk. 1% and 0.3% monophase collagen scaffolds and Integra® disks served as controls. Mechanical characterization as well as in vitro and in vivo invasion assays were performed. Cell number and depth of invasion were analyzed using Imaris™. Cell identity was assessed immunohistochemically. RESULTS: In vitro, MSS exhibited significantly greater average depth of cellular invasion than Integra® and monophase collagen controls. MSS also demonstrated significantly higher cell counts than controls. In vivo, MSS revealed significantly more cellular invasion spanning the entire scaffold depth at 14 days than Integra®. CD31+ expressing luminal structures suggestive of neovasculature were seen within MSS at 7 days and were more prevalent after 14 days. Multiphoton microscopy of MSS demonstrated erythrocytes within luminal structures after 14 days. CONCLUSION: By harnessing simple architectural cues to induce cellular migration, MSS holds great potential for clinical translation as the next generation dermal replacement product. STATEMENT OF SIGNIFICANCE: Large skin wounds require tissue engineered dermal substitutes in order to promote healing. Currently available dermal replacement products do not always adequately incorporate into the body, especially in complex wounds, due to poor neovascularization. In this paper, we present a hydrogel with an innovative microarchitecture that is composed of dense type I collagen microspheres suspended in a less-dense collagen bulk. We show that cell invasion into the scaffold is driven solely by mechanical cues inherent within this differential density interface, and that this induces robust vascular cell invasion both in vitro and in a rodent model. Our hydrogel performs favorably compared to the current clinical gold standard, Integra®. We believe this hydrogel scaffold may be the first of the next generation of dermal replacement products.

Fabrication of capillary-like structures with Pluronic F127® and Kerria lacca resin (shellac) in biocompatible tissue-engineered constructs.

The fabrication of large cellular tissue-engineered constructs is currently limited by an inability to manufacture internal vasculature that can be anastomosed to the host circulatory system. Creation of synthetic tissues with microvascular networks that adequately mimic the size and density of in vivo capillaries remains one of the foremost challenges within tissue engineering, as cells must reside within 200-300 µm of vasculature for long-term survival. In our previous work, we used a sacrificial microfibre technique whereby Pluronic® F127 fibres were embedded and then sacrificed within a collagen matrix, leaving behind a patent channel, which was subsequently seeded with endothelial and smooth muscle cells, forming a neointima and neomedia. We now have extended our technique and describe two approaches to synthesize a biocompatible tissue-engineered construct with macro-inlet and -outlet vessels, bridged by a dense network of cellularized microvessels, recapitulating the hierarchical organization of an arteriole, venule and capillary bed, respectively. Copyright © 2016 John Wiley & Sons, Ltd.

Optimizing cell sourcing for clinical translation of tissue engineered ears.

Background . Currently, the major impediment to clinical translation of our previously described platform for the fabrication of high fidelity, patient-specific tissue engineered ears is the development of a clinically optimal cell sourcing strategy. A limited autologous auricular chondrocyte (AuC) supply in conjunction with rapid chondrocyte de-differentiation during in vitro expansion currently makes clinical translation more challenging. Mesenchymal stem cells (MSCs) offer significant promise due to their inherent chondrogenic potential, and large availability through minimally invasive procedures. Herein, we demonstrate the promise of AuC/MSC co-culture to fabricate elastic cartilage using 50% fewer AuC than standard approaches. METHODS: Bovine auricular chondrocytes (bAuC) and bovine MSC (bMSC) were encapsulated within 10 mg ml-1 type I collagen hydrogels in ratios of bAuC:bMSC 100:0, 50:50, and 0:100 at a density of 25 million cells ml-1 hydrogel. One mm thick collagen sheet gels were fabricated, and thereafter, 8 mm diameter discs were extracted using a biopsy punch. Discs were implanted subcutaneously in the dorsa of nude mice (NU/NU) and harvested after 1 and 3 months. RESULTS: Gross analysis of explanted discs revealed bAuC:bMSC co-culture discs maintained their size and shape, and exhibited native auricular cartilage-like elasticity after 1 and 3 months of implantation. Co-culture discs developed into auricular cartilage, with viable chondrocytes within lacunae, copious proteoglycan and elastic fiber deposition, and a distinct perichondrial layer. Biochemical analysis confirmed that co-culture discs deposited critical cartilage molecular components more readily than did both bAuC and bMSC discs after 1 and 3 months, and proteoglycan content significantly increased between 1 and 3 months. CONCLUSION: We have successfully demonstrated an innovative cell sourcing strategy that facilitates our efforts to achieve clinical translation of our high fidelity, patient-specific ears for auricular reconstruction utilizing only half of the requisite auricular chondrocytes to fabricate mature elastic cartilage.

A Novel Three-Dimensional Platform to Investigate Neoangiogenesis, Transendothelial Migration, and Metastasis of MDAMB-231 Breast Cancer Cells.

BACKGROUND: A crucial step in the progression of cancer involves the transendothelial migration of tumor cells into the bloodstream and invasion at distant sites. Most in vitro models of malignant cell behavior do not account for the presence of and interaction with vascular cells. Three-dimensional platforms to further explore the factors responsible for metastatic cellular behavior are under intensive investigation. METHODS: Hydrogels with encapsulated MDAMB-231 breast cancer cells were fabricated with a central microchannel. The microchannel was lined with a co-culture of human umbilical vein endothelial cells and human aortic smooth muscle cells. For comparison, co-culture-seeded microchannels without breast cancer cells (MDAMB-negative) were fabricated. RESULTS: After 7 and 14 days, the endoluminal lining of encapsulated MDAMB-231 co-culture-seeded microchannels demonstrated aberrant endothelial cell and smooth muscle cell organization and breast cancer cell transendothelial migration. MDAMB-231 cells performed matrix remodeling, forming tumor aggregates within the bulk, migrating preferentially toward the hydrogel "neovessel." In contrast, MDAMB-negative constructs demonstrated maintenance of an intact endoluminal lining composed of endothelial cells and smooth muscle cells that organized into discrete layers. Furthermore, the thicknesses of the endoluminal lining of MDAMB-negative constructs were significantly greater than encapsulated MDAMB-231 co-culture-seeded constructs after 7 and 14 days (p = 0.012 and p < 0.001, respectively). CONCLUSION: The authors have created a powerful tool that may have tremendous impact on furthering our understanding of cancer recurrence and metastasis, shedding light on these poorly understood phenomena.

Long-Term Morphological and Microarchitectural Stability of Tissue-Engineered, Patient-Specific Auricles In Vivo.

Current techniques for autologous auricular reconstruction produce substandard ear morphologies with high levels of donor-site morbidity, whereas alloplastic implants demonstrate poor biocompatibility. Tissue engineering, in combination with noninvasive digital photogrammetry and computer-assisted design/computer-aided manufacturing technology, offers an alternative method of auricular reconstruction. Using this method, patient-specific ears composed of collagen scaffolds and auricular chondrocytes have generated auricular cartilage with great fidelity following 3 months of subcutaneous implantation, however, this short time frame may not portend long-term tissue stability. We hypothesized that constructs developed using this technique would undergo continued auricular cartilage maturation without degradation during long-term (6 month) implantation. Full-sized, juvenile human ear constructs were injection molded from high-density collagen hydrogels encapsulating juvenile bovine auricular chondrocytes and implanted subcutaneously on the backs of nude rats for 6 months. Upon explantation, constructs retained overall patient morphology and displayed no evidence of tissue necrosis. Limited contraction occurred in vivo, however, no significant change in size was observed beyond 1 month. Constructs at 6 months showed distinct auricular cartilage microstructure, featuring a self-assembled perichondrial layer, a proteoglycan-rich bulk, and rounded cellular lacunae. Verhoeff's staining also revealed a developing elastin network comparable to native tissue. Biochemical measurements for DNA, glycosaminoglycan, and hydroxyproline content and mechanical properties of aggregate modulus and hydraulic permeability showed engineered tissue to be similar to native cartilage at 6 months. Patient-specific auricular constructs demonstrated long-term stability and increased cartilage tissue development during extended implantation, and offer a potential tissue-engineered solution for the future of auricular reconstructions.