Supercritical carbon dioxide extracted extracellular matrix material from adipose tissue.

Adipose tissue is a rich source of extracellular matrix (ECM) material that can be isolated by delipidating and decellularizing the tissue. However, the current delipidation and decellularization methods either involve tedious and lengthy processes or require toxic chemicals, which may result in the elimination of vital proteins and growth factors found in the ECM. Hence, an alternative delipidation and decellularization method for adipose tissue was developed using supercritical carbon dioxide (SC-CO2) that eliminates the need of any harsh chemicals and also reduces the amount of processing time required. The resultant SC-CO2-treated ECM material showed an absence of nuclear content but the preservation of key proteins such as collagen Type I, collagen Type III, collagen Type IV, elastin, fibronectin and laminin. In addition, other biological factors such as glycosaminoglycans (GAGs) and growth factors such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) were also retained. Subsequently, the resulting SC-CO2-treated ECM material was used as a bioactive coating on tissue culture plastic (TCP). Four different cell types including adipose tissue-derived mesenchymal stem cells (ASCs), human umbilical vein endothelial cells (HUVECs), immortalized human keratinocyte (HaCaT) cells and human monocytic leukemia cells (THP-1) were used in this study to show that the SC-CO2-treated ECM coating can be potentially used for various biomedical applications. The SC-CO2-treated ECM material showed improved cell-material interactions for all cell types tested. In addition, in vitro scratch wound assay using HaCaT cells showed that the presence of SC-CO2-treated ECM material enhanced keratinocyte migration whilst the in vitro cellular studies using THP-1-derived macrophages showed that the SC-CO2-treated ECM material did not evoke pro-inflammatory responses from the THP-1-derived macrophages. Overall, this study shows the efficacy of SC-CO2 method for delipidation and decellularization of adipose tissue whilst retaining its ECM and its subsequent utilization as a bioactive surface coating material for soft tissue engineering, angiogenesis and wound healing applications.

Culturing fibroblasts in 3D human hair keratin hydrogels.

Human hair keratins are readily available, easy to extract, and eco-friendly materials with natural bioactivities. Keratin-based materials have been studied for applications such as cell culture substrates, internal hemostats for liver injury, and conduits for peripheral nerve repair. However, there are limited reports of using keratin-based 3D scaffolds for cell culture in vitro. Here, we describe the development of a 3D hair keratin hydrogel, which allows for living cell encapsulation under near physiological conditions. The convenience of making the hydrogels from keratin solutions in a simple and controllable manner is demonstrated, giving rise to constructs with tunable physical properties. This keratin hydrogel is comparable to collagen hydrogels in supporting the viability and proliferation of L929 murine fibroblasts. Notably, the keratin hydrogels contract less significantly as compared to the collagen hydrogels, over a 16-day culture period. In addition, preliminary in vivo studies in immunocompetent animals show mild acute host tissue response. These results collectively demonstrate the potential of cell-loaded keratin hydrogels as 3D cell culture systems, which may be developed for clinically relevant applications.

Early controlled release of peroxisome proliferator-activated receptor ß/δ agonist GW501516 improves diabetic wound healing through redox modulation of wound microenvironment.

Diabetic wounds are imbued with an early excessive and protracted reactive oxygen species production. Despite the studies supporting PPARß/δ as a valuable pharmacologic wound-healing target, the therapeutic potential of PPARß/δ agonist GW501516 (GW) as a wound healing drug was never investigated. Using topical application of polymer-encapsulated GW, we revealed that different drug release profiles can significantly influence the therapeutic efficacy of GW and consequently diabetic wound closure. We showed that double-layer encapsulated GW microparticles (PLLA:PLGA:GW) provided an earlier and sustained dose of GW to the wound and reduced the oxidative wound microenvironment to accelerate healing, in contrast to single-layered PLLA:GW microparticles. The underlying mechanism involved an early GW-mediated activation of PPARß/δ that stimulated GPx1 and catalase expression in fibroblasts. GPx1 and catalase scavenged excessive H2O2 accumulation in diabetic wound beds, prevented H2O2-induced ECM modification and facilitated keratinocyte migration. The microparticles with early and sustained rate of GW release had better therapeutic wound healing activity. The present study underscores the importance of drug release kinetics on the therapeutic efficacy of the drug and warrants investigations to better appreciate the full potential of controlled drug release.

From flab to fab: transforming surgical waste into an effective bioactive coating material.

Cellular events are regulated by the interaction between integrin receptors in the cell membrane and the extracellular matrix (ECM). Hence, ECM, as a material, can potentially play an instructive role in cell-material interactions. Currently, adipose tissue in the form of lipoaspirate is often discarded. Here, it is demonstrated how our chemical-free decellularization method could be used to obtain ECM from human lipoaspirate waste material. These investigations show that the main biological components are retained in the lipoaspirate-derived ECM (LpECM) material and that this LpECM material could subsequently be used as a coating material to confer bioactivity to an otherwise inert biodegradable material (i.e., polycaprolactone). Overall, lipoaspirate material, a complex blend of endogenous proteins, is effectively used a bioactive coating material. This work is an important stepping-stone towards the development of biohybrid scaffolds that contain cellular benefits without requiring the use of additional biologics based on commonly discarded lipoaspirate material.

Angiopoietin-like 4 stimulates STAT3-mediated iNOS expression and enhances angiogenesis to accelerate wound healing in diabetic mice.

Impaired wound healing is a major source of morbidity in diabetic patients. Poor outcome has, in part, been related to increased inflammation, poor angiogenesis, and deficiencies in extracellular matrix components. Despite the enormous impact of these chronic wounds, effective therapies are lacking. Here, we showed that the topical application of recombinant matricellular protein angiopoietin-like 4 (ANGPTL4) accelerated wound reepithelialization in diabetic mice, in part, by improving angiogenesis. ANGPTL4 expression is markedly elevated upon normal wound injury. In contrast, ANGPTL4 expression remains low throughout the healing period in diabetic wounds. Exogenous ANGPTL4 modulated several regulatory networks involved in cell migration, angiogenesis, and inflammation, as evidenced by an altered gene expression signature. ANGPTL4 influenced the expression profile of endothelial-specific CD31 in diabetic wounds, returning its profile to that observed in wild-type wounds. We showed ANGPTL4-induced nitric oxide production through an integrin/JAK/STAT3-mediated upregulation of inducible nitric oxide synthase (iNOS) expression in wound epithelia, thus revealing a hitherto unknown mechanism by which ANGPTL4 regulated angiogenesis via keratinocyte-to-endothelial-cell communication. These data show that the replacement of ANGPTL4 may be an effective adjunctive or new therapeutic avenue for treating poor healing wounds. The present finding also confirms that therapeutic angiogenesis remains an attractive treatment modality for diabetic wound healing.

Evaluation of sustained release of PLC-loaded prednisolone acetate microfilm on postoperative inflammation in an experimental model of glaucoma filtration surgery.

PURPOSE: To evaluate the effect of a biodegradable microfilm with sustained release of prednisolone acetate (PA) on postoperative wound healing after experimental glaucoma filtration surgery (GFS). METHODS: Biodegradable microfilms composed of poly (D-, L-lactide-co-caprolactone) (PLC) were fabricated and then pre-loaded PA-20% total weight. Fourteen New Zealand White rabbits were randomly divided into 3 treatment groups: GFS alone (n=4), GFS with PLC microfilms (n=4) and GFS with PA-loaded microfilm (n=6). Microfilms were inserted subconjunctivally, adjacent to the filtering surgical site. We monitored all eyes with slit-lamp examination, bleb photography and anterior segment optical coherence tomography (AS-OCT). Histology with immunohistochemistry was performed to determine the presence of any inflammation. RESULTS: Prednisolone acetate 20%-loaded microfilms exhibited steady, sustained release in vitro. Eyes implanted with PA-loaded microfilms showed a significantly better bleb survival (100% vs. 37.5%, p<0.001) and reduced bleb vascularity (58%; 95% CI 54-62% vs. 30%; 95% CI 23-37%, p=0.001) compared to the control at 30 days postoperatively. Histology and immunohistochemistry demonstrated less T-cell infiltration in the eye implanted with PA-loaded microfilms. CONCLUSION: Subconjunctival insertion of a PA-loaded biodegradeable microfilm exhibit sustained release of PA to reduce postoperative inflammation and prolong bleb survival in rabbit GFS.

Sustained release of an anti-glaucoma drug: demonstration of efficacy of a liposomal formulation in the rabbit eye.

Topical medication remains the first line treatment of glaucoma; however, sustained ocular drug delivery via topical administration is difficult to achieve. Most drugs have poor penetration due to the multiple physiological barriers of the eye and are rapidly cleared if applied topically. Currently, daily topical administration for lowering the intra-ocular pressure (IOP), has many limitations, such as poor patient compliance and ocular allergy from repeated drug administration. Poor compliance leads to suboptimal control of IOP and disease progression with eventual blindness. The delivery of drugs in a sustained manner could provide the patient with a more attractive alternative by providing optimal therapeutic dosing, with minimal local toxicity and inconvenience. To investigate this, we incorporated latanoprost into LUVs (large unilamellar vesicles) derived from the liposome of DPPC (di-palmitoyl-phosphatidyl-choline) by the film hydration technique. Relatively high amounts of drug could be incorporated into this vesicle, and the drug resides predominantly in the bilayer. Vesicle stability monitored by size measurement and DSC (differential scanning calorimetry) analysis showed that formulations with a drug/lipid mole ratio of about 10% have good physical stability during storage and release. This formulation demonstrated sustained release of latanoprost in vitro, and then tested for efficacy in 23 rabbits. Subconjunctival injection and topical eye drop administration of the latanoprost/liposomal formulation were compared with conventional daily administration of latanoprost eye drops. The IOP lowering effect with a single subconjunctival injection was shown to be sustained for up to 50 days, and the extent of IOP lowering was comparable to daily eye drop administration. Toxicity and localized inflammation were not observed in any treatment groups. We believe that this is the first demonstration, in vivo, of sustained delivery to the anterior segment of the eye that is safe and efficacious for 50 days.

Biocompatibility and biodegradation studies of subconjunctival implants in rabbit eyes.

Sustained ocular drug delivery is difficult to achieve. Most drugs have poor penetration due to the multiple physiological barriers of the eye and are rapidly cleared if applied topically. Biodegradable subconjunctival implants with controlled drug release may circumvent these two problems. In our study, two microfilms (poly [d,l-lactide-co-glycolide] PLGA and poly[d,l-lactide-co-caprolactone] PLC were developed and evaluated for their degradation behavior in vitro and in vivo. We also evaluated the biocompatibility of both microfilms. Eighteen eyes (9 rabbits) were surgically implanted with one type of microfilm in each eye. Serial anterior-segment optical coherence tomography (AS-OCT) scans together with serial slit-lamp microscopy allowed us to measure thickness and cross-sectional area of the microfilms. In vitro studies revealed bulk degradation kinetics for both microfilms, while in vivo studies demonstrated surface erosion kinetics. Serial slit-lamp microscopy revealed no significant inflammation or vascularization in both types of implants (mean increase in vascularity grade PLGA50/50 12±0.5% vs. PLC70/30 15±0.6%; P = 0.91) over a period of 6 months. Histology, immunohistochemistry and immuno-fluorescence also revealed no significant inflammatory reaction from either of the microfilms, which confirmed that both microfilms are biocompatible. The duration of the drug delivery can be tailored by selecting the materials, which have different degradation kinetics, to suit the desired clinical therapeutic application.