Nanofiber matrix formulations for the delivery of Exendin-4 for tendon regeneration: In vitro and in vivo assessment.

Tendon and ligament injuries are the most common musculoskeletal injuries, which not only impact the quality of life but result in a massive economic burden. Surgical interventions for tendon/ligament injuries utilize biological and/or engineered grafts to reconstruct damaged tissue, but these have limitations. Engineered matrices confer superior physicochemical properties over biological grafts but lack desirable bioactivity to promote tissue healing. While incorporating drugs can enhance bioactivity, large matrix surface areas and hydrophobicity can lead to uncontrolled burst release and/or incomplete release due to binding. To overcome these limitations, we evaluated the delivery of a peptide growth factor (exendin-4; Ex-4) using an enhanced nanofiber matrix in a tendon injury model. To overcome drug surface binding due to matrix hydrophobicity of poly(caprolactone) (PCL)-which would be expected to enhance cell-material interactions-we blended PCL and cellulose acetate (CA) and electrospun nanofiber matrices with fiber diameters ranging from 600 to 1000 nm. To avoid burst release and protect the drug, we encapsulated Ex-4 in the open lumen of halloysite nanotubes (HNTs), sealed the HNT tube endings with a polymer blend, and mixed Ex-4-loaded HNTs into the polymer mixture before electrospinning. This reduced burst release from ∼75% to ∼40%, but did not alter matrix morphology, fiber diameter, or tensile properties. We evaluated the bioactivity of the Ex-4 nanofiber formulation by culturing human mesenchymal stem cells (hMSCs) on matrix surfaces for 21 days and measuring tenogenic differentiation, compared with nanofiber matrices in basal media alone. Strikingly, we observed that Ex-4 nanofiber matrices accelerated the hMSC proliferation rate and elevated levels of sulfated glycosaminoglycan, tendon-related genes (Scx, Mkx, and Tnmd), and ECM-related genes (Col-I, Col-III, and Dcn), compared to control. We then assessed the safety and efficacy of Ex-4 nanofiber matrices in a full-thickness rat Achilles tendon defect with histology, marker expression, functional walking track analysis, and mechanical testing. Our analysis confirmed that Ex-4 nanofiber matrices enhanced tendon healing and reduced fibrocartilage formation versus nanofiber matrices alone. These findings implicate Ex-4 as a potentially valuable tool for tendon tissue engineering.

Natural Polymer-Based Micronanostructured Scaffolds for Bone Tissue Engineering.

Although bone tissue allografts and autografts aremoften used as a regenerative tissue during the bone healing, their availability, donor site morbidity, and immune response to grafted tissue are limiting factors their more common usage. Tissue engineered implants, such as acellular or cellular polymeric structures, can be an alternative solution. A variety of scaffold fabrication techniques including electrospinning, particulate leaching, particle sintering, and more recently 3D printing have been used to create scaffolds with interconnected pores and mechanical properties for tissue regeneration. Simply combining particle sintering and molecular self-assembly to create porous microstructures with imbued nanofibers to produce micronanostructures for tissue regeneration applications. Natural polymers like polysaccharides, proteins and peptides of plant or animal origin have gained significant attention due to their assured biocompatibility in tissue regeneration. However, majority of these polymers are water soluble and structures derived from them are in the form of hydrogels and require additional stabilization via cross-linking. For bone healing applications scaffolds are required to be strong, and support attachment, proliferation and differentiation of osteoprogenitors into osteoblasts. Our ongoing work utilizes plant polysaccharide cellulose derivatives and collagen to create mechanically stable and bioactive micronanostructured scaffold for bone tissue engineering. Scaffold microstructure is essentially solvent sintered cellulose acetate (CA) microspheres in the form of a negative template for trabecular bone with defined pore and mechanical properties. Collagen nanostructures are imbued into the 3D environment of CA scaffolds using collagen molecular self-assembly principles. The resultant CA-collagen micronanostructures provide the benefits of combined polymers and serve as an alternative material platform to many FDA approved polyesters. Our ongoing studies and published work confirm improved osteoprogenitor adhesion, proliferation, migration, differentiation, extracellular matrix (ECM) secretion in promoting bone healing. In this chapter we will provide a detailed protocol on the creation of micronanostructured CA-collagen scaffolds and their characterization for bone tissue engineering using human mesenchymal stem cells.

Assessment of Hedgehog Signaling Pathway Activation for Craniofacial Bone Regeneration in a Critical-Sized Rat Mandibular Defect.

IMPORTANCE: Osseous craniofacial defects are currently reconstructed with bone grafting, rigid fixation, free tissue transfer, and/or recombinant human bone morphogenetic protein 2. Although these treatment options often have good outcomes, they are associated with substantial morbidity, and many patients are not candidates for free tissue transfer. OBJECTIVE: To assess whether polysaccharide-based scaffold (PS) constructs that are cross-linked with smoothened agonist (SAG), vascular endothelial growth factor (VEGF), and bone morphogenetic protein 6 (BMP-6) would substantially increase bone regeneration. DESIGN, SETTING, AND PARTICIPANTS: This animal model study was conducted at the University of Virginia School of Medicine Cui Laboratory from March 1, 2017, to June 30, 2017. Thirty-three 10-week-old female Lewis rats were acquired for the study. Bilateral nonsegmental critical-sized defects were created in the angle of rat mandibles. The defects were either left untreated or filled with 1 of the 9 PSs. The rats were killed after 8 weeks, and bone regeneration was evaluated using microcomputed tomographic imaging and mechanical testing. Analysis of variance testing was used to compare the treatment groups. MAIN OUTCOMES AND MEASURES: Blinded analysis and computer analysis of the microcomputed tomographic images were used to assess bone regeneration. RESULTS: In the 33 female Lewis rats, minimal healing was observed in the untreated mandibles. Addition of SAG was associated with increases in bone regeneration and bone density in all treatment groups, and maximum bone healing was seen in the group with BMP-6, VEGF, and SAG cross-linked to PS. For each of the 5 no scaffold group vs BMP-6, VEGF, and SAG cross-linked to PS group comparisons, mean defect bone regeneration was 4.14% (95% CI, 0.94%-7.33%) vs 66.19% (95% CI, 54.47%-77.90%); mean bone volume, 14.52 mm3 (95% CI, 13.07-15.97 mm3) vs 20.87 mm3 (95% CI, 14.73- 27.01 mm3); mean bone surface, 68.97 mm2 (95% CI, 60.08-77.85 mm2) vs 96.77 mm2 (95% CI, 76.11-117.43 mm2); mean ratio of bone volume to total volume, 0.11 (95% CI, 0.10-0.11) vs 0.15 (95% CI, 0.10-0.19); and mean connectivity density 0.03 (95% CI, 0.02-0.05) vs 0.32 (95% CI, 0.25-0.38). On mechanical testing, mandibles with untreated defects broke with less force than control mandibles in which no defect was made, although this force did not reach statistical significance. No significant difference in force to fracture was observed among the treatment groups. CONCLUSIONS AND RELEVANCE: In this rat model study, activation of the hedgehog signaling pathway using smoothened agonist was associated with increased craniofacial bone regeneration compared with growth factors alone, including US Food and Drug Administration-approved recombinant human bone morphogenetic protein 2. Pharmaceuticals that target this pathway may offer a new reconstructive option for bony craniofacial defects as well as nonunion and delayed healing fractures. LEVEL OF EVIDENCE: NA.

Micro-Nanostructures of Cellulose-Collagen for Critical Sized Bone Defect Healing.

Bone tissue engineering strategies utilize biodegradable polymeric matrices alone or in combination with cells and factors to provide mechanical support to bone, while promoting cell proliferation, differentiation, and tissue ingrowth. The performance of mechanically competent, micro-nanostructured polymeric matrices, in combination with bone marrow stromal cells (BMSCs), is evaluated in a critical sized bone defect. Cellulose acetate (CA) is used to fabricate a porous microstructured matrix. Type I collagen is then allowed to self-assemble on these microstructures to create a natural polymer-based, micro-nanostructured matrix (CAc). Poly (lactic-co-glycolic acid) matrices with identical microstructures serve as controls. Significantly higher number of implanted host cells are distributed in the natural polymer based micro-nanostructures with greater bone density and more uniform cell distribution. Additionally, a twofold increase in collagen content is observed with natural polymer based scaffolds. This study establishes the benefits of natural polymer derived micro-nanostructures in combination with donor derived BMSCs to repair and regenerate critical sized bone defects. Natural polymer based materials with mechanically competent micro-nanostructures may serve as an alternative material platform for bone regeneration.

Prognostic indices of perioperative outcome following transperitoneal laparoscopic adrenalectomy.

OBJECTIVES: We sought to identify preoperative patient and tumor characteristics that may be useful prognostic indicators of postsurgical outcome in patients undergoing laparoscopic adrenalectomy (LA). SUBJECTS AND METHODS: Data from 92 patients who underwent 93 transabdominal LA procedures between 2006-2012 were retrieved. Patients were stratified based on estimated blood loss (EBL), length of stay (LOS), and perioperative complications. Interdependencies between surgical outcome and patient demographics, tumor characteristics, comorbidities, and Charlson Comorbidity Index (CCI) were statistically analyzed. The predictive capacity of each index was assessed using receiver operating characteristic curves. RESULTS: Neither age, gender, tumor laterality, body mass index, American Society of Anesthesiologists (ASA) score, nor CCI predicted the occurrence of perioperative complications. EBL was significantly associated with increased age, tumor size, ASA score, and CCI, whereas prolonged LOS was associated with higher ASA score. Tumor size was related, although not significantly, to LOS and perioperative complications. Tumors ≥7.5 cm in diameter were significantly associated with worse perioperative outcomes. CONCLUSIONS: LA for adrenal lesions demonstrated reasonable complication rates and perioperative outcomes. Tumor size, CCI, and ASA score are predictive of increased EBL and LOS.

Osteoinductive small molecules: growth factor alternatives for bone tissue engineering.

Tissue engineering aims to repair, restore, and regenerate lost or damaged tissues by using biomaterials, cells, mechanical forces and factors (chemical and biological) alone or in combination. Growth factors are routinely used in the tissue engineering approach to expedite the process of regeneration. The growth factor approach has been hampered by several complications including high dose requirements, lower half-life, protein instability, higher costs and undesired side effects. Recently a variety of alternative small molecules of both natural and synthetic origin have been explored as alternatives to growth factors for tissue regeneration applications. Small molecules are simple biochemical components that elicit certain cellular responses through signaling cascades. Small molecules present a viable alternative to biological factors. Small molecule strategies can reduce various side effects, maintain bioactivity in a biological environment and minimize cost issues associated with complex biological growth factors. This manuscript focuses on three-osteoinductive small molecules, namely melatonin, resveratrol (from natural sources) and purmorphamine (synthetically designed) as inducers of bone formation and osteogenic differentiation of stem cells. Efforts have been made to summarize possible biological pathways involved in the action of each of these drugs. Melatonin is known to affect Mitogen Activated Protein (MAP) kinase, Bone morphogenic protein (BMP) and canonical wnt signaling. Resveratrol is known to activate cascades involving Wnt and NAD-dependent deacetylase sirtuin-1 (Sirt1). Purmorphamine is a Hedgehog (Hh) pathway agonist as it acts on Smoothened (Smo) receptors. These mechanisms and the way they are affected by the respective small molecules will also be discussed in the manuscript.

Cellulose and collagen derived micro-nano structured scaffolds for bone tissue engineering.

Scaffold based bone tissue engineering (BTE) has made great progress in regenerating lost bone tissue. Materials of natural and synthetic origin have been used for scaffold fabrication. Scaffolds derived from natural polymers offer greater bioactivity and biocompatibility with mammalian tissues to favor tissue healing, due to their similarity to native extracellular matrix (ECM) components. Often it is a challenge to fabricate natural polymer based scaffolds for BTE applications without compromising their bioactivity, while maintaining adequate mechanical properties. In this work, we report the fabrication and characterization of cellulose and collagen based micro-nano structured scaffolds using human osteoblasts (HOB) for BTE applications. These porous micro-nano structured scaffolds (average pore diameter 190 +/- 10 microm) exhibited mechanical properties in the mid range of human trabecular bone (compressive modulus 266.75 +/- 33.22 MPa and strength 12.15 3 +/- 2.23 MPa). These scaffolds supported the greater adhesion and phenotype maintenance of cultured HOB as reflected by higher levels of osteogenic enzyme alkaline phosphatase and mineral deposition compared to control polyester micro-nano structured scaffolds of identical pore properties. These natural polymer based micro-nano structured scaffolds may serve as alternatives to polyester based scaffolds for BTE applications.