Pilot Clinical Evaluation of PoreSkin: A Human Acellular Dermal Matrix in Burn Scars.

Background: An extensive full-thickness wound need a graft, sometime very large. However, donor sites are often limited. Dermal substitutes are among the tissue-engineered products applied to clinical use. PoreSkin, a human acellular dermal matrix (hADM) manufactured by the Faculty of Medicine, Chulalongkorn University, is the first human dermal substitute developed in Thailand. Objective: Assess the safety and ability in achieving durable and definitively cosmetic coverage using PoreSkin. Material and Method: Eleven hypertrophic burn scars were enrolled in the present study. After scar excision, PoreSkin was placed followed by delayed split-thickness skin graft, three weeks later. The primary outcomes were the engraftment rate of the Poreskin and the skin graft. The secondary outcomes included complications and the final cosmetic appearance. Results: The engraftment rate of PoreSkin was 97.7% at day 21. The engraftment rate of autologous sheet skin graft placed over PoreSkin was 91.8%. Regarding the quality of the scar, using the Vancouver scar scale, it shows a statistically significant improvement (p<0.05). No major complications or rejection were observed. Conclusion: The performance of PoreSkin as a human acellular dermal matrix (hADM) is comparable to other commercial dermal substitutes in term of engraftment rate, complications, and rejection.

Development of collagen/demineralized bone powder scaffolds and periosteum-derived cells for bone tissue engineering application.

The aim of this study was to investigate physical and biological properties of collagen (COL) and demineralized bone powder (DBP) scaffolds for bone tissue engineering. DBP was prepared and divided into three groups, based on various particle sizes: 75-125 µm, 125-250 µm, and 250-500 µm. DBP was homogeneously mixed with type I collagen and three-dimensional scaffolds were constructed, applying chemical crosslinking and lyophilization. Upon culture with human periosteum-derived cells (PD cells), osteogenic differentiation of PD cells was investigated using alkaline phosphatase (ALP) activity and calcium assay kits. The physical properties of the COL/DBP scaffolds were obviously different from COL scaffolds, irrespective of the size of DBP. In addition, PD cells cultured with COL scaffolds showed significantly higher cell adhesion and proliferation than those with COL/DBP scaffolds. In contrast, COL/DBP scaffolds exhibited greater osteoinductive potential than COL scaffolds. The PD cells with COL/DBP scaffolds possessed higher ALP activity than those with COL scaffolds. PD cells cultured with COL/DBP scaffolds with 250-500 mm particle size yielded the maximum calcium deposition. In conclusion, PD cells cultured on the scaffolds could exhibit osteoinductive potential. The composite scaffold of COL/DBP with 250-500 mm particle size could be considered a potential bone tissue engineering implant.

Acanthus ebracteatus Vahl. ethanol extract enhancement of the efficacy of the collagen scaffold in wound closure: a study in a full-thickness-wound mouse model.

Acanthus ebracteatus Vahl. is a Thai herb that is effective in wound healing. We sought to quantitatively determine whether or not the combined application of Acanthus ebracteatus Vahl. and a collagen scaffold will increase wound closure and angiogenesis. Balb/c mice (body weight: 22-25 g) were anesthetized with sodium thiopental. The dorsal skin incision measuring 1.5 × 1.5 cm was made and then deepened using scissors to produce a full-thickness incision down to the level of the panniculus carnosus. The size of the wound was approximately 10% of the total body surface area. The collagen sheet was implanted onto the wound. Animals were divided into 4 major groups as follows: wound with normal saline (W-NSS), wound treated with 0.3 g/kg BW of Acanthus ebracteatus Vahl. extract (W-AE (0.3 g/kg.bw)), wound implanted with collagen scaffold (W-Coll), and wound implanted with collagen scaffold and treated with 0.3 g/kg BW of Acanthus ebracteatus Vahl. (W-Coll-AE combination). On day 14, the W-Coll-AE group showed decreased wound areas and increased capillary vascularity (CV) when compared to the other 3 groups, W-NSS, W-AE0.3, and W-Coll. In the present study, the combination of AE0.3 with collagen showed the best effect on skin angiogenesis and promoted wound closure with less neutrophil infiltration.

Tissue response and biodegradation of composite scaffolds prepared from Thai silk fibroin, gelatin and hydroxyapatite.

This work aimed to investigate tissue responses and biodegradation, both in vitro and in vivo, of four types of Bombyx mori Thai silk fibroin based-scaffolds. Thai silk fibroin (SF), conjugated gelatin/Thai silk fibroin (CGSF), hydroxyapatite/Thai silk fibroin (SF4), and hydroxyapatite/conjugated gelatin/Thai silk fibroin (CGSF4) scaffolds were fabricated using salt-porogen leaching, dehydrothermal/chemical crosslinking and an alternate soaking technique for mineralization. In vitro biodegradation in collagenase showed that CGSF scaffolds had the slowest biodegradability, due to the double crosslinking by dehydrothermal and chemical treatments. The hydroxyapatite deposited from alternate soaking separated from the surface of the protein scaffolds when immersed in collagenase. From in vivo biodegradation studies, all scaffolds could still be observed after 12 weeks of implantation in subcutaneous tissue of Wistar rats and also following ISO10993-6: Biological evaluation of medical devices. At 2 and 4 weeks of implantation the four types of Thai silk fibroin based-scaffolds were classified as "non-irritant" to "slight-irritant", compared to Gelfoam(®) (control samples). These natural Thai silk fibroin-based scaffolds may provide suitable biomaterials for clinical applications.

Immobilization of biomolecules on the surface of electrospun polycaprolactone fibrous scaffolds for tissue engineering.

To make polycaprolactone (PCL) more suitable for tissue engineering, PCL in the form of electrospun fibrous scaffolds was first modified with 1,6-hexamethylenediamine to introduce amino groups on their surface. Various biomolecules, i.e., collagen, chitosan, and Gly-Arg-Gly-Asp-Ser (GRGDS) peptide, were then immobilized on their surface, with N,N'-disuccinimidylcarbonate being used as the coupling agent. Dynamic water contact angle measurement indicated that the scaffold surface became more hydrophilic after the aminolytic treatment and the subsequent immobilization of the biomolecules. The appropriateness of these PCL fibrous scaffolds for the tissue/cell culture was evaluated in vitro with three different cell lines, e.g., mouse fibroblasts (L929), human epidermal keratinocytes (HEK001), and mouse calvaria-derived preosteoblastic cells (MC3T3-E1). Both the neat and the modified PCL fibrous scaffolds released no substances in the levels that were harmful to these cells. Among the various biomolecule-immobilized PCL fibrous scaffolds, the ones that had been immobilized with type I collagen, a Arg-Gly-Asp-containing protein, showed the greatest ability to support both the attachment and the proliferation of all of the investigated cell types, followed by those that had been immobilized with GRGDS peptide.

Development of acellular dermis from porcine skin using periodic pressurized technique.

In this work, a new method for producing acellular dermis (ADM), a natural scaffold used for dermal replacement, from porcine skin was developed. Fresh porcine skin from local slaughterhouse was dehaired by sodium sulphide following by epidermis removal using glycerol. After fat removal by chloroform/methanol (2/1 v/v) solvent, cellular components were removed using enzymatic treatment incorporated with a periodic pressurized technique. The effects of enzyme type (trypsin and dispase II) and periodic pressurized conditions on the efficiency of cell removal were investigated. When periodic pressure was applied, enzymatic treatment time could be shorten since the enzyme solution was able to penetrate into tight dermis. As a result, cells could be easily removed from porcine skin as noticed quantitatively by DNA assay and qualitatively by H&E staining. When enzyme refreshment was introduced into the decellularized process, the percentage of cell removal was further enhanced. This ensured that no inhibitions effect from the removed cells on enzyme-substrate interaction. Moreover, short-time enzymatic treatment with periodic pressurized technique could prevent the disruption of dermal structure, as observed by SEM. Dispase II can be used to remove cell better than trypsin in the periodic pressurized technique. However, in vivo study indicated that numerous fibroblast from the host tissue infiltrated into ADM prepared using both enzymes. Neo-collagen and neo-capillaries were produced in both implanted ADMs. The result elucidated that the use of periodic pressurized technique with enzymatic treatment has a high potential to be a new method to produce ADM for skin tissue engineering.

In vitro biocompatibility of electrospun poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) fiber mats.

In the present contribution, the potential for use of the ultrafine electrospun fiber mats of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) as scaffolding materials for skin and nerve regeneration was evaluated in vitro using mouse fibroblasts (L929) and Schwann cells (RT4-D6P2T) as reference cell lines. Comparison was made with PHB and PHBV films that were prepared by solution-casting technique. Indirect cytotoxicity assessment of the as-spun PHB and PHBV fiber mats with mouse fibroblasts (L929) and Schwann cells (RT4-D6P2T) indicated that the materials were acceptable to both types of cells. The attachment of L929 on all of the fibrous scaffolds was significantly better than that on both the film scaffolds and tissue-culture polystyrene plate (TCPS), while RT4-D6P2T appeared to attach on the flat surfaces of TCPS and the film scaffolds much better than on the rough surfaces of the fibrous scaffolds. For L929, all of the fibrous scaffolds were superior in supporting the cell proliferation to the film counterparts, but inferior to TCPS at days 3 and 5, while, for RT4-D6P2T, the rough surfaces of the fibrous scaffolds appeared to be very poor in supporting the cell proliferation when comparing with the smooth surfaces of TCPS and the film scaffolds. Scanning electron microscopy was also used to observe the behavior of both types of cells that were cultured on both the fibrous and the film scaffolds and glass substrate for 24 h.

Tissue engineering of cartilage with porous polycarprolactone--alginate scaffold: the first report of tissue engineering in Thailand.

OBJECTIVE: To engineer human cartilage with porous polycaprolactone (PCL)-Alginate Scaffold. BACKGROUND: Polycaprolactone (PCL) is a prolonged degradable polymer that has good mechanical strength. The authors fabricated PCL as an ear shaped scaffold. Alginate hydrogel was used to seed chondrocyte into the PCL porous scaffold by a gel-cell seeding technique. MATERIAL AND METHOD: PCL Scaffolds were fabricated like human pinna by particle leaching technique. Chondrocyte was isolated from human rib cartilage and then cultured. The cultured chondrocyte were mixed with 1.2% alginate and b-FGF (basic-fibroblast growth factor) 5 ng/ml at a concentration of 25 x 10(6) cell/ml, then were seeded in porous PCL scaffold to make the constructs. The constructs were cultured in vitro for 1 week. Then they were implanted in subcutaneous plane of the back of six-female nude mice (5 weeks old). Two nude mice were sacrificed at 2, 3, and 6 months. Histological study was done (H&E, Alcian blue, collagen type II). RESULT: Neocartilage was formed in the porous cavity of PCL scaffold. At 2 and 3 months, neocartilage were similar to very young cartilage. At 6 months, they were mature. The delayed maturation until 6 months and the highly vascularization of neocartilage in the early phase was the effect of human b-FGF The growths of neocartilage islands in porous cavity were also observed along with degradation ofPCL inter-porous septum. CONCLUSION: This paper reports the first success of cartilage tissue engineering in Thailand.

Tissue engineered muscle implantation for tongue reconstruction: a preliminary report.

OBJECTIVES/HYPOTHESIS: Because current tongue reconstructive methods introduce adynamic, variably sensate tissue into the mouth, the critical functions of the tongue in articulation and deglutition may be compromised. The objective of this work was to introduce a combination of myoblasts and scaffolding material into rat hemiglossectomy defects and to examine the extent of neomuscle formation in the reconstructed area, under the hypothesis that the presence of myoblasts leads to formation of new muscle. STUDY DESIGN: Randomized, prospective animal study. METHODS: Myoblasts were harvested from neonatal Lewis rats, and a growth factor enriched collagen gel was prepared. Syngeneic adult animals received either hemiglossectomy alone or reconstruction with one of four experimental reconstructive preparations: collagen gel alone, collagen gel with suspended myoblasts, the gel-cell combination in undifferentiated muscle construct form by way of tissue culture for 7 days in a preformed mold, or differentiated constructs, cultured in myoblast fusion medium. After 6 or 16 weeks, animal weight gain was recorded, animals were killed, and the tongues harvested. The tissue was examined histologically, and quality of the muscular regenerate was rated on a scale according to predefined criteria. RESULTS: Animals in all groups gained weight appropriately. In groups receiving hemiglossectomy alone or acellular (gel only) reconstruction, there was significant scarring and lack of neomuscle formation. In groups receiving myoblast transplantation, either by way of gel suspension or in the form of undifferentiated or differentiated constructs, muscle quality was superior to controls. CONCLUSIONS: Myoblast transplantation into hemiglossectomy defects appears to lead to new muscle formation and does not inhibit normal weight gain in animals after tongue implantation.

Prospective, randomized, controlled trial of proximally based vs. distally based gluteus maximus flap for anal incontinence in cadavers.

PURPOSE: The aim of this study was to compare the lengths of proximally based and distally based gluteus maximus flaps created as for anal sphincter reconstruction in soft human cadavers. METHODS: Twelve soft cadavers were used in this prospective, randomized, controlled study. In each cadaver, a proximally based flap of the gluteus maximus muscle was performed on one side and a distally based flap on the other. All flaps were carefully dissected with neurovascular preservation by one surgeon. After the dissected flap was placed across the anus, the length of the flap that projected beyond the anus was measured. The differences between such lengths of both types of flaps were assessed by paired t-test. RESULTS: The average lengths of the parts that projected beyond the anus for proximally based and distally based flaps were 8.08 and 4.50 (standard deviation, 0.51 and 0.79) cm, respectively. The average difference was 3.58 (standard deviation, 0.51) cm, which was statistically significant (P < 0.001). CONCLUSION: Results showed that proximally based flaps were significantly longer than distally based flaps and that transposition and wrapping around the anus with proximally based flaps were always easily performed without tension. These findings support the use of unilateral proximally based gluteus maximus flaps instead of unilateral or bilateral distally based flaps in patients with anal incontinence. The location of the neurovascular pedicle of the gluteus maximus was consistent at 1 cm superior and lateral to ischial tuberosity. Knowledge of this landmark allows quick and safe dissection of the gluteus maximus flap.