Rachel C. Hooper

Reduction of suture associated inflammation after 28 days using novel biocompatible pseudoprotein poly(ester amide) biomaterials

Sutures elicit an inflammatory response, which may impede the healing process and result in wound complications. We recently reported a novel family of biocompatible, biodegradable polymers, amino acid-based poly(ester amide)s (AA-PEA), which we have shown to significantly attenuate the foreign body inflammatory response in vitro. Two types of AA-PEA (Phe-PEA and Arg-Phe-PEA) were used to coat silk or plain-gut sutures, which were implanted in the gluteus muscle of C57BL/6 mice, while the uncoated control sutures were implanted in the contralateral side. After 3, 7, 14, and 28 days the mean area of inflammation surrounding the sutures was compared. Phe-PEA coating of silk sutures significantly decreased inflammation compared with noncoated controls (67.8u2009±u200917.4% after 3d [pu2009=u20090.0014], 51.6u2009±u20097.2% after 7d [pu2009<u20090.001], and 37.3u2009±u20098.3% after 28d [pu2009=u20090.0001]) when assessed via analysis of photomicrographs using digital image software. Phe-PEA coated plain-gut sutures were similarly assessed and demonstrated a significant decrease in the mean area of inflammation across all time points (54.1u2009±u20098.3% after 3 d, 41.4u2009±u20093.9% after 7 d, 71.5u2009±u20098.1% after 14 d, 78.4u2009±u20098.5%, and after 28 d [all pu2009<u20090.0001]). Arg-Phe-PEA coated silk demonstrated significantly less inflammation compared to noncoated controls (61.3u2009±u20099.4% after 3 d, 44.7u2009±u20094.7% after 7 d, 19.6u2009±u20098%, and 38.3u2009±u20096.8% after 28 d [all pu2009<u20090.0001]), as did coated plain-gut (37.4u2009±u20098.3% after 3 d [pu2009=u20090.0004], 55.0u2009±u20097.8% after 7 d [pu2009<u20090.0001], 46.0u2009±u20094.6% after 14 d [pu2009<u20090.0001], and 59.0u2009±u20097.9% after 28 d [pu2009<u20090.0001]). Both Phe-PEA and Arg-Phe-PEA coatings significantly decrease the inflammatory response to sutures in vivo for up to 28 days.

Vascular smooth muscle cell optimization of vasculogenesis within naturally derived, biodegradable, hybrid hydrogel scaffolds.

Background: As vascularization represents the rate-limiting step in permanent incorporation of hydrogel-based tissue-regeneration templates, the authors sought to identify the material chemistry that would optimize endothelial cell adhesion and invasion into custom hydrogel constructs. The authors further investigated induction of endothelial tubule formation by growth factor supplementation and paracrine stimulation. Methods: Hydrogel scaffolds consisting of combinations of alginate, collagen type I, and chitosan were seeded with human umbilical vein endothelial cells and maintained under standard conditions for 14 days. Cell density and invasion were then evaluated. Tubule formation was evaluated following basic fibroblast growth factor addition or co-culture with human aortic smooth muscle cells. Results: Human umbilical vein endothelial cells demonstrated greatest cell-surface density and invasion volumes with alginate and collagen (10:1 weight/weight) scaffolds (p < 0.05). Supplementation with basic fibroblast growth factor increased surface density but neither invasion nor tubule formation. A significant increase in tubule content/organization was observed with increasing human aortic smooth muscle cell–to–human umbilical vein endothelial cell ratio co-culture. Conclusions: Alginate and collagen 10:1 scaffolds allow for maximal cellularization compared with other combinations studied. Growth factor supplementation did not affect human umbilical vein endothelial cell invasion or morphology. Paracrine signaling by means of co-culture with human umbilical vein endothelial cells stimulated endothelial tubule formation and vascular protonetwork organization. These findings serve to guide future endeavors toward fabrication of prevascularized tissue constructs.

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. Verhoeffs 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.

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

Abstract 162: in vivo microanastomosis of microvessel containing tissue-engineered constructs: the final frontier.

PurPose: Although autologous tissue transfer has been established as a reliable approach to the reconstruction of complex defects, there are associated consequences including donor site pain, functional loss, paresthesias, dysthesthia, and scarring. The ability to synthesize vascularized constructs for the management of these complex wounds would represent a quantum leap in the field of tissue engineering. In previous work we synthesized and performed an in vivo microvascular anastomosis of a collagen construct containing an unseeded internal longitudinal microchannel with inlet and outlet. Here we fabricate and microsurgically anastomose collagen constructs containing an internal endothelialized microchannel.

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.

Abstract 94: Patient-Specific Tissue Engineered Constructs for Ear Reconstruction

PURPOSE: The current gold standard for reconstruction of pediatric microtia is autologous costal cartilage reconstruction. This method is limited due to morbidity at the donor site, failure to match auricular cartilage properties, and difficulty accurately recreating the ear morphology of the individual patient. We have previously demonstrated the capacity to fabricate high fidelity patient-specific ear constructs using digital photogrammetry and CAD/CAM techniques, and that these constructs formed auricular cartilage when implanted in vivo for up to 3 months. We have now applied the same methods for constructs implanted in vivo for 6 months.

Abstract P29: Optimization of Functional, Perfusable Vascular Networks within Tissue Engineered Hydrogels

eleCtroPhysiology: SSEP negative peak amplitudes were significantly decreased (p<.05) at POD 14, 28, 37, and 79 when compared to baseline with significant recovery at POD 79. SSEP stimulation thresholds were significantly increased (p<.05) at POD 37 and 79 with significant recovery occurring at POD 79. Abductor pollicis brevis thresholds obtained from Tc-MEP stimulation were significantly increased at POD 37 and 79. CNAPs were first measured at POD 42 and confirmed with stimulation-response curve. Nerve conduction velocity was 40% baseline at POD 90 with significantly increased CNAP stimulation thresholds (p<.05).

Abstract 50: fabrication of tissue-engineered human constructs for patient specific auricles.

PurPose: The reconstruction of pediatric microtia using autologous donor cartilage is limited by significant obligatory donor site, pain and scarring as well as frequent suboptimal aesthetic outcome. Tissue-engineering allows for the creation of anatomically correct auricular constructs and the minimization or even elimination of the previously mentioned complications. In previous work, we fabricated patient specific, high fidelity tissue-engineered frameworks composed of type I collagen and bovine auricular chondrocytes that not only maintained shape and size over 12 weeks, but also exhibited proteoglycan and elastin deposition, with mechanical properties indistinguishable from native auricular cartilage. As a bridge to clinical translation, we have now synthesized human auricular chondrocyte (HAC) constructs in order to determine the optimal chrondrocyte passage and seeding density for the fabrication of patient specific tissue-engineered auricles.

Abstract P1: Bioprinted Vascularized Tissue-Engineered Constructs for In Vivo Perfusion

PurPose: The greatest challenge to contemporary tissue engineers remains the difficulty associated with creating vascular networks within the engineered tissue. Furthermore, any vascularized construct must be designed to allow for anastomosis to the host vascular system. In previous work we synthesized a tissue-engineered scaffold containing an endothelialized internal loop microchannel for microsurgical anastomosis and in vivo perfusion utilizing a sacrificial microfiber technique. Bioprinting is an emerging technology that allows for the consistent and rapid fabrication of three-dimensional constructs comprised of any combination of extrudable polymers and cells with a precise predetermined microarchitecture. Here we describe the fabrication of bioprinted hydrogel constructs for cell seeding and in vivo microanastomosis.