Design and biofabrication of dermal regeneration scaffolds: role of oligomeric collagen fibril density and architecture

Aim: To evaluate dermal regeneration scaffolds custom-fabricated from fibril-forming oligomeric collagen where the total content and spatial gradient of collagen fibrils was specified. Materials & methods: Microstructural and mechanical features were verified by electron microscopy and tensile testing. The ability of dermal scaffolds to induce regeneration of rat full-thickness skin wounds was determined and compared with no fill control, autograft skin and a commercial collagen dressing. Results: Increasing fibril content of oligomer scaffolds inhibited wound contraction and decreased myofibroblast marker expression. Cellular and vascular infiltration of scaffolds over the 14-day period varied with the graded density and orientation of fibrils. Conclusion: Fibril content, spatial gradient and orientation are important collagen scaffold design considerations for promoting vascularization and dermal regeneration while reducing wound contraction

In Situ Type I Oligomeric Collagen Macroencapsulation Promotes Islet Longevity and Function in Vitro and in Vivo

Widespread use of pancreatic islet transplantation for treatment of type 1 diabetes (T1D) is currently limited by requirements for long-term immunosuppression, limited donor supply, and poor long-term engraftment and function. Upon isolation from their native microenvironment, islets undergo rapid apoptosis, which is further exacerbated by poor oxygen and nutrient supply following infusion into the portal vein. Identifying alternative strategies to restore critical microenvironmental cues, while maximizing islet health and function, is needed to advance this cellular therapy. We hypothesized that biophysical properties provided through type I oligomeric collagen macroencapsulation are important considerations when designing strategies to improve islet survival, phenotype, and function. Mouse islets were encapsulated at various Oligomer concentrations (0.5–3.0 mg/ml) or suspended in media and cultured for 14 days, after which viability, protein expression, and function were assessed. Oligomer-encapsulated islets showed a density-dependent improvement in in vitro viability, cytoarchitecture, and insulin secretion, with 3 mg/ml yielding values comparable to freshly isolated islets. For transplantation into streptozotocin-induced diabetic mice, 500 islets were mixed in Oligomer and injected subcutaneously, where rapid in situ macroencapsulation occurred, or injected with saline. Mice treated with Oligomer-encapsulated islets exhibited rapid (within 24 h) diabetes reversal and maintenance of normoglycemia for 14 (immunocompromised), 90 (syngeneic), and 40 days (allogeneic). Histological analysis showed Oligomer-islet engraftment with maintenance of islet cytoarchitecture, revascularization, and no foreign body response. Oligomer-islet macroencapsulation may provide a useful strategy for prolonging the health and function of cultured islets and has potential as a subcutaneous injectable islet transplantation strategy for treatment of T1D

Regenerative tissue filler for breast conserving surgery and other soft tissue restoration and reconstruction needs

Complete removal of cancerous tissue and preservation of breast cosmesis with a single breast conserving surgery (BCS) is essential for surgeons. New and better options would allow them to more consistently achieve this goal and expand the number of women that receive this preferred therapy, while minimizing the need for re-excision and revision procedures or more aggressive surgical approaches (i.e., mastectomy). We have developed and evaluated a regenerative tissue filler that is applied as a liquid to defects during BCS prior to transitioning to a fibrillar collagen scaffold with soft tissue consistency. Using a porcine simulated BCS model, the collagen filler was shown to induce a regenerative healing response, characterized by rapid cellularization, vascularization, and progressive breast tissue neogenesis, including adipose tissue and mammary glands and ducts. Unlike conventional biomaterials, no foreign body response or inflammatory-mediated “active” biodegradation was observed. The collagen filler also did not compromise simulated surgical re-excision, radiography, or ultrasonography procedures, features that are important for clinical translation. When post-BCS radiation was applied, the collagen filler and its associated tissue response were largely similar to non-irradiated conditions; however, as expected, healing was modestly slower. This in situ scaffold-forming collagen is easy to apply, conforms to patient-specific defects, and regenerates complex soft tissues in the absence of inflammation. It has significant translational potential as the first regenerative tissue filler for BCS as well as other soft tissue restoration and reconstruction needs

Amine functionalization of cholecyst-derived extracellular matrix with generation 1 PAMAM dendrimer

This document is the unedited author's version of a Submitted Work that was subsequently accepted for publication in Biomacromolecules, copyright © American Chemical Society after peer review. To access the final edited and published work, see http://pubs.acs.org/doi/pdf/10.1021/bm701055k.A method to functionalize cholecyst-derived extracellular matrix (CEM) with free amine groups was established in an attempt to improve its potential for tethering of bioactive molecules. CEM was incorporated with Generation-1 polyamidoamine (G1 PAMAM) dendrimer by using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide and N-hydroxysuccinimide cross-linking system. The nature of incorporation of PAMAM dendrimer was evaluated using shrink temperature measurements, Fourier transform infrared (FTIR) assessment, ninhydrin assay, and swellability. The effects of PAMAM incorporation on mechanical and degradation properties of CEM were evaluated using a uniaxial mechanical test and collagenase degradation assay, respectively. Ninhydrin assay and FTIR assessment confirmed the presence of increasing free amine groups with increasing quantity of PAMAM in dendrimer-incorporated CEM (DENCEM) scaffolds. The amount of dendrimer used was found to be critical in controlling scaffold degradation, shrink temperature, and free amine content. Cell culture studies showed that fibroblasts seeded on DENCEM maintained their metabolic activity and ability to proliferate in vitro. In addition, fluorescence cell staining and scanning electron microscopy analysis of cell-seeded DENCEM showed preservation of normal fibroblast morphology and phenotype

Quantitative Analysis of the Effect of Cancer Invasiveness and Collagen Concentration on 3D Matrix Remodeling

Extracellular matrix (ECM) remodeling is a key component of cell migration and tumor metastasis, and has been associated with cancer progression. Despite the importance of matrix remodeling, systematic and quantitative studies on the process have largely been lacking. Furthermore, it remains unclear if the disrupted tensional homeostasis characteristic of malignancy is due to initially altered ECM and tissue properties, or to the alteration of the tissue by tumor cells. To explore these questions, we studied matrix remodeling by two different prostate cancer cell lines in a three-dimensional collagen system. Over one week, we monitored structural changes in gels of varying collagen content using confocal reflection microscopy and quantitative image analysis, tracking metrics of fibril fraction, pore size, and fiber length and diameter. Gels that were seeded with no cells (control), LNCaP cells, and DU-145 cells were quantitatively compared. Gels with higher collagen content initially had smaller pore sizes and higher fibril fractions, as expected. However, over time, LNCaP- and DU-145-populated matrices showed different structural properties compared both to each other and to the control gels, with LNCaP cells appearing to favor microenvironments with lower collagen fiber fractions and larger pores than DU-145 cells. We posit that the DU-145 cells' preference for denser matrices is due to their higher invasiveness and proteolytic capabilities. Inhibition of matrix proteases resulted in reduced fibril fractions for high concentration gels seeded with either cell type, supporting our hypothesis. Our novel quantitative results probe the dynamics of gel remodeling in three dimensions and suggest that prostate cancer cells remodel their ECM in a synergistic manner that is dependent on both initial matrix properties as well as their invasiveness

Functional tissue engineering of ligament healing

Ligaments and tendons are dense connective tissues that are important in transmitting forces and facilitate joint articulation in the musculoskeletal system. Their injury frequency is high especially for those that are functional important, like the anterior cruciate ligament (ACL) and medial collateral ligament (MCL) of the knee as well as the glenohumeral ligaments and the rotator cuff tendons of the shoulder. Because the healing responses are different in these ligaments and tendons after injury, the consequences and treatments are tissue- and site-specific. In this review, we will elaborate on the injuries of the knee ligaments as well as using functional tissue engineering (FTE) approaches to improve their healing. Specifically, the ACL of knee has limited capability to heal, and results of non-surgical management of its midsubstance rupture have been poor. Consequently, surgical reconstruction of the ACL is regularly performed to gain knee stability. However, the long-term results are not satisfactory besides the numerous complications accompanied with the surgeries. With the rapid development of FTE, there is a renewed interest in revisiting ACL healing. Approaches such as using growth factors, stem cells and scaffolds have been widely investigated. In this article, the biology of normal and healing ligaments is first reviewed, followed by a discussion on the issues related to the treatment of ACL injuries. Afterwards, current promising FTE methods are presented for the treatment of ligament injuries, including the use of growth factors, gene delivery, and cell therapy with a particular emphasis on the use of ECM bioscaffolds. The challenging areas are listed in the future direction that suggests where collection of energy could be placed in order to restore the injured ligaments and tendons structurally and functionally

Impairment of Human Neutrophil Oxidative Burst By

We report evidence of a novel mechanism by which polychlorinated biphenyls might act as potent inducers of inflammation. Aroclor 1242 (A1242), a polychlorinated biphenyl mixture, and 2,2',4,4'-tetrachlorobiphenyl (PCB47), a constituent of A1242 that produces the same patterns of effects, impaired the oxidative burst of human neutrophils by inhibiting the antioxidant enzyme superoxide dismutase, which converts O 2 to H 2 O 2 . Pre-incubation of neutrophils with A1242 or PCB47 before stimulation with phorbol 12-myristate 13-acetate heightened the respiratory burst, producing a significant increase in intracellular production along with a significant decrease in H 2 O 2 production compared with unexposed agoniststimulated neutrophils. This was also evident in a physiologically relevant situation in which neutrophils pre-incubated with A1242 were subsequently stimulated with a combination of N-formyl-L- methionyl-L-leucyl-L-phenylalanine and tumor necrosis factor-a. Incubation of bovine copper-zinc superoxide dismutase (EC 1.15.1.1) with A1242 or PCB47 in a cell-free system reversed the enzymemediated inhibition of 6-hydroxydopamine autoxidation, indicating that polychlorinated biphenyls inhibited superoxide dismutase activity. Low superoxide dismutase activity in neutrophils leads to imbalances between production of free radicals and antioxidant defense mechanisms, which can in turn induce tissue damage and hasten the onset of neutrophil apoptosis. J. Leukoc. Biol. 63: 216-- 224; 1998