Marcus Thien Chong Wong

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.

Early controlled release of peroxisome proliferator-activated receptor β/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 PPARβ/δ as a valuable pharmacologic wound-healing target, the therapeutic potential of PPARβ/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 PPARβ/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.

Bioactivated protein-based porous microcarriers for tissue engineering applications

Microcarriers are commonly used in tissue engineering applications as they provide a large surface area for cell attachment. However, limited research has been done on ovalbumin (OVA), which is a relatively cheap protein found in avian egg white. Hence, in our current study OVA is fabricated into porous microcarriers and the effect of different OVA to alginate ratios on the properties of OVA microcarriers was investigated. Subsequently, in order to further improve cell-material interactions, the extracellular matrix (ECM) material isolated from the human lipoaspirate material was conjugated with the porous OVA microcarriers using carbodiimide chemistry. A waste-to-resource strategy was employed to obtain this ECM material from the human lipoaspirate material, which typically is discarded after surgery. This study illustrates the possibility of obtaining ECM material using a physical decellularization method as well as the novel application of ECM material as a coating to confer bioactivity to protein-based microcarriers such as OVA. The incorporation of lipoaspirate-derived ECM (LpECM) into the OVA microstructure has been shown to improve mechanical strength and promote cellular growth on the microcarriers. The resulting porous OVA-LpECM hybrid microcarriers with tunable mechanical properties are examples of bioactivated porous protein-based microcarriers that can be applied in the field of tissue engineering.

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.

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.

Three Dimensional Printing of Titanium for Bone Tissue Engineering Applications: A Preliminary Study

One of the main goals of bone tissue engineering is the development of scaffolds that mimic both functional and structural properties of native bone itself. This study describes the preliminary work carried out to assess the viability of using three dimensional printing (3DP) technology for the fabrication of porous titanium scaffolds with lowered modulus and improved biocompatibility. 3DP enables the manufacturing of three dimensional (3D) objects with a defined structure directly from a Computer Aided Design (CAD). The overall porosity of the 3D structures is contributed by the presence of both pores-by-process (PBP) and pores-by-design (PBD). This study mainly focuses on the PBP, which are formed during the sintering step as the result of the removal of the binding agent polyvinyl alcohol (PVA). Sintering temperatures of 1250oC, 1350oC and 1370oC were used during the fabrication process. Our results showed that by varying the binder percentage and the sintering temperature, pores with diameters in the range of approximately 17-24 μm could be reproducibly achieved. Other physical properties such as surface roughness, porosity and average pore size were also measured for all sample groups. Results from subsequent cell culture studies using adipose tissue-derived mesenchymal stem cells (ASCs) showed improved attachment, viability and proliferation for the 3DP titanium samples as compared to the two-dimensional (2D) dense titanium samples. Hence, based on our current preliminary studies, 3DP technology can potentially be used to fabricate customized, patient-specific metallic bone implants with lowered modulus. This can effectively help in prevention of stress-shielding, and enhancement of implant fixation in vivo. It is envisioned that an optimized combination of binder percentage and sintering temperature can result in the fabrication of scaffolds with the desired porosity and mechanical properties to fit the intended clinical application.

A novel application of calcium phosphate-based bone cement as an adjunct procedure in adult craniofacial reconstruction.

Secondary corrective osteotomy of malunited craniofacial fractures can be a challenging proposition. The exposure, extrusion, and palpability of the titanium implants used become a genuine concern especially in areas of relatively thin skin, such as the periorbital region. Restoring a satisfactory contour to the midface is another major task for the plastic surgeon. Bone cement used to reconstruct craniofacial defects has existed for many years. However, most applications have been as a substitute for autogenous bone grafts for defects less than 25 cm2. In this article, we present two cases of malunited facial fractures that underwent corrective osteotomy, during which we felt that despite the conventional osteotomy and reduction techniques, there was still either a small remnant step deformity or suboptimal contour smoothness due to prominence of the implants used. We thus used bone cement as a resurfacing medium over titanium implants to restore good malar contour and reduce the palpability and exposure rate of the titanium implants. We report good patient satisfaction with contour correction with no increase in wound infection rates or any delay in wound healing. There was initial chemosis associated with the use of the bone cement, which resolved in both patients within 3 to 4 weeks. Postoperative computed tomography showed some degree of osteointegration but no fraction of the bone cement. Calcium phosphate bone cement thus presents an attractive adjunctive method for midfacial contour resurfacing, when used in conjunction with conventional osteotomy procedures and as an onlay over prominent titanium implants.

Angiopoietin-like 4 induces a β-catenin-mediated upregulation of ID3 in fibroblasts to reduce scar collagen expression

In adult skin wounds, collagen expression rapidly re-establishes the skin barrier, although the resultant scar is aesthetically and functionally inferior to unwounded tissue. Although TGFβ signaling and fibroblasts are known to be responsible for scar-associated collagen production, there are currently no prophylactic treatments for scar management. Fibroblasts in crosstalk with wound keratinocytes orchestrate collagen expression, although the precise paracrine pathways involved remain poorly understood. Herein, we showed that the matricellular protein, angiopoietin-like 4 (ANGPTL4), accelerated wound closure and reduced collagen expression in diabetic and ANGPTL4-knockout mice. Similar observations were made in wild-type rat wounds. Using human fibroblasts as a preclinical model for mechanistic studies, we systematically elucidated that ANGPTL4 binds to cadherin-11, releasing membrane-bound β-catenin which translocate to the nucleus and transcriptionally upregulate the expression of Inhibitor of DNA-binding/differentiation protein 3 (ID3). ID3 interacts with scleraxis, a basic helix-loop-helix transcription factor, to inhibit scar-associated collagen types 1α2 and 3α1 production by fibroblasts. We also showed ANGPTL4 interaction with cadherin-11 in human scar tissue. Our findings highlight a central role for matricellular proteins such as ANGPTL4 in the attenuation of collagen expression and may have a broader implication for other fibrotic pathologies.

Primary mucinous carcinoma (PMC) of the skin—a case report

Primary mucinous carcinoma of the skin is a rare malignant adnexal neoplasm. It was first described by Lennox et al. in 1952. It is uncertain whether it is of apocrine or eccrine origin (Wright and Font, Cancer 44:1757–1768, 2; Mendoza and Helwig, Arch Dermatol 103:68–78, 3). Showing morphological similarity to mucinous carcinoma of the breast, it may be difficult to distinguish it from a cutaneous metastatic deposit of mucinous adenocarcinoma from a visceral primary lesion. Hence, it deserves attention not only for its scarcity but also for the challenge in determining its precise diagnosis and hence, planning its management. We present a case of primary mucinous carcinoma of the skin.

Case series: pedicled vertical rectus myocutaneous flaps for cover of breast and perineal defects—a 2-year review in our centre

Colorectal carcinoma is the most common cancer afflicting both Singaporean men and women; whereas, breast carcinoma is the most frequent female-specific cancer according to the report published by the Singapore Cancer Registry on cancer trends from 2003 to 2007. As such, breast and perineal defects requiring soft tissue cover is a challenging occurrence after initial tumour surgery by the breast and colorectal surgeons, especially for fungating breast tumours and very low colorectal carcinomas, respectively. In Tan Tock Seng Hospital prior to 2007, many of these patients may have had their wounds skin-grafted or allowed to heal by secondary intention. This often involved prolonged hospital stay, difficult wound care and significant morbidity as well as a negative impact on the quality of life for these patients. Pedicled vertical rectus myocutaneous (VRAM) flaps were then introduced for cover of these defects as an effective method of reducing these negative aspects of patient care. This case series aims to review the efficacy of the VRAM flap in achieving this within a 2-year period. Over this period, the VRAM flap has shown to decrease length of hospital stay, reduction of the duration to commencement of adjuvant therapy, and also decrease in the difficulty of wound care as well as the complication rate.