Qixu Zhang

Combining decellularized human adipose tissue extracellular matrix and adipose-derived stem cells for adipose tissue engineering

Repair of soft tissue defects resulting from lumpectomy or mastectomy has become an important rehabilitation process for breast cancer patients. This study aimed to provide an adipose tissue engineering platform for soft tissue defect repair by combining decellularized human adipose tissue extracellular matrix (hDAM) and human adipose-derived stem cells (hASCs). To derive hDAM incised human adipose tissues underwent a decellularization process. Effective cell removal and lipid removal were proved by immunohistochemical analysis and DNA quantification. Scanning electron microscopic examination showed a three-dimensional nanofibrous architecture in hDAM. The hDAM included collagen, sulfated glycosaminoglycan, and vascular endothelial growth factor, but lacked major histocompatibility complex antigen I. hASC viability and proliferation on hDAM were proven in vitro. hDAM implanted subcutaneously in Fischer rats did not cause an immunogenic response, and it underwent remodeling, as indicated by host cell infiltration, neovascularization, and adipose tissue formation. Fresh fat grafts (Coleman technique) and engineered fat grafts (hDAM combined with hASCs) were implanted subcutaneously in nude rats. The implanted engineered fat grafts maintained their volume for 8 weeks, and the hASCs contributed to adipose tissue formation. In summary, the combination of hDAM and hASCs provides not only a clinically translatable platform for adipose tissue engineering, but also a vehicle for elucidating fat grafting mechanisms.

Decellularized tracheal matrix scaffold for tissue engineering

Background: A tracheal matrix scaffold decellularized by detergent-enzymatic treatment has been shown as a promising scaffold in tracheal tissue engineering. The objectives of this study were to evaluate the impact of this technique on tracheal extracellular matrix integrity and characterize the matrix environment for recellularization. Methods: Brown Norway rat tracheae were decellularized using a modified detergent-enzymatic treatment. Antigenicity and cellularity were monitored during processing. Glycosaminoglycan content, histoarchitecture, and mechanical properties were also evaluated. Matrix compatibility was determined by cytotoxicity assay. Surface ultrastructure of the matrix and its interaction with seeded bone marrow stem cell–derived chondrocytes and tracheal epithelial cells were examined by scanning electron microscopy. Results: Rat trachea treated with five detergent-enzymatic treatment cycles demonstrated complete elimination of antigenicity. Although there was a significant loss of glycosaminoglycan (t test, p < 0.01), histoarchitecture of tracheal cartilage and basement membrane was retained after decellularization. Stiffness decreased, but sufficient compressive strength was preserved to maintain lumen patency. The decellularized matrix showed good cell compatibility and favored adhesion and growth of chondrocytes and respiratory epithelial cells, as demonstrated by scanning electron microscopy. Conclusions: At the point of complete antigen removal, detergent-enzymatic treatment altered tracheal extracellular matrix composition but preserved the major structure and adequate mechanical strength. The matrix provided a compatible and supportive environment for recellularization.

Perichondrium directed cartilage formation in silk fibroin and chitosan blend scaffolds for tracheal transplantation.

The purpose of this study was to investigate the potential of silk fibroin and chitosan blend (SFCS) biological scaffolds for the purpose of cartilage tissue engineering with applications in tracheal tissue reconstruction. The capability of these scaffolds as cell carrier systems for chondrocytes was determined in vitro and cartilage generation in vivo on engineered chondrocyte-scaffold constructs with and without a perichondrium wrapping was tested in an in vivo nude mouse model. SFCS scaffolds supported chondrocyte adhesion, proliferation, and differentiation, determined as features of the cells based on the spherical cell morphology, increased accumulation of glycosaminoglycans, and increased collagen type II deposition with time within the scaffold framework. Perichondrium wrapping significantly (P<0.001) improved chondrogenesis within the cell-scaffold constructs in vivo. In vivo implantation for 6weeks did not generate cartilage structures resembling native trachea, although cartilage-like structures were present. The mechanical properties of the regenerated tissue increased due to the deposition of chondrogenic matrix within the SFCS scaffold structural framework of the trachea. The support of chondrogenesis by the SFCS tubular scaffold construct resulted in a mechanically sound structure and thus is a step towards an engineered trachea that could potentially support the growth of an epithelial lining resulting in a tracheal transplant with properties resembling those of the fully functional native trachea.

Engineering vascularized soft tissue flaps in an animal model using human adipose-derived stem cells and VEGF+PLGA/PEG microspheres on a collagen-chitosan scaffold with a flow-through vascular pedicle

Insufficient neovascularization is associated with high levels of resorption and necrosis in autologous and engineered fat grafts. We tested the hypothesis that incorporating angiogenic growth factor into a scaffold-stem cell construct and implanting this construct around a vascular pedicle improves neovascularization and adipogenesis for engineering soft tissue flaps. Poly(lactic-co-glycolic-acid/polyethylene glycol (PLGA/PEG) microspheres containing vascular endothelial growth factor (VEGF) were impregnated into collagen-chitosan scaffolds seeded with human adipose-derived stem cells (hASCs). This setup was analyzed in vitro and then implanted into isolated chambers around a discrete vascular pedicle in nude rats. Engineered tissue samples within the chambers were harvested and analyzed for differences in vascularization and adipose tissue growth. In vitro testing showed that the collagen-chitosan scaffold provided a supportive environment for hASC integration and proliferation. PLGA/PEG microspheres with slow-release VEGF had no negative effect on cell survival in collagen-chitosan scaffolds. In vivo, the system resulted in a statistically significant increase in neovascularization that in turn led to a significant increase in adipose tissue persistence after 8 weeks versus control constructs. These data indicate that our model-hASCs integrated with a collagen-chitosan scaffold incorporated with VEGF-containing PLGA/PEG microspheres supported by a predominant vascular vessel inside a chamber-provides a promising, clinically translatable platform for engineering vascularized soft tissue flap. The engineered adipose tissue with a vascular pedicle could conceivably be transferred as a vascularized soft tissue pedicle flap or free flap to a recipient site for the repair of soft-tissue defects.

Clinical application of the anterolateral thigh flap for soft tissue reconstruction.

The purpose of this article is to describe the authors experience using the anterolateral thigh (ALT) flap for the reconstruction of a variety of soft tissue defects. The flap utility and donor site morbidity were evaluated in 126 cases from March 1985 to August 2007. The ALT flaps were harvested as either free fasciocutaneous, free adipofascial, fasciocutaneous island, or reversed fasciocutaneous island flaps to repair facial, neck, breast, trunk, and extremity defects. In 40 cases (32%), the skin vessels were found to be septocutaneous perforators, and in 86 cases (68%), they were found as musculocutaneous perforators. Of the 126 flaps, 121 survived completely, providing a success rate of 96.0%. There were four cases undergoing multidetector-row computed tomographic angiography (CTA) for preoperative perforator mapping, and all perforators were confirmed intraoperatively. In conclusion, the ALT flap is a versatile and reliable flap that could well be a priority option for soft tissue reconstruction. CTA can provide more valuable and accurate anatomic information about the pedicle and perforators, making it safer and faster to harvest a targeted ALT perforator flap with less donor site morbidity.

Decellularized tracheal matrix scaffold for tracheal tissue engineering: in vivo host response.

Background: The authors have previously demonstrated promising results with tissue engineered trachea in vitro using decellularized matrix scaffolds. The present study aims to investigate the applicability of the construct in vivo. Methods: Tracheae harvested from Brown Norway rats (donor) and Lewis rats (recipient) were decellularized with repeated detergent-enzymatic treatment cycles. Decellularized Brown Norway tracheal matrix scaffolds were seeded with Lewis rat stem cell–derived chondrocytes externally and tracheal epithelial cells internally to generate a bilaminated tracheal construct. Brown Norway tracheal matrix scaffolds (n = 6), Lewis rat scaffolds (n = 6), and the engineered constructs (n = 3) were implanted subcutaneously in Lewis rats and observed for 4 weeks. Fresh Brown Norway (n = 6) and Lewis rat (n = 6) tracheae were implanted as controls. Histologic analysis for macrophage, CD8, and CD4 cell infiltration was performed. Results: Allogeneic decellularized matrix scaffold showed significantly decreased macrophage, CD8+ and CD4+ cell infiltration compared with tracheal allografts, and demonstrated similar level of cell infiltration to syngeneic decellularized matrix scaffold. No significant differences in macrophage infiltration were observed between syngeneic decellularized matrix scaffolds and tracheal isografts. The engineered constructs achieved complete epithelial cell coverage and preserved lumen patency; however, chondrocytes failed to repopulate the cartilaginous matrix with statically seeding stem cell on scaffold. Conclusions: Decellularized tracheal matrix scaffold did not induce significant allograft rejection or foreign body reaction in vivo. Although the construct supported reepithelialization, stem cell–derived chondrocytes failed to engraft in the heterotopic environment and represent a focus of future investigations.

Harvesting Technique Affects Adipose-Derived Stem Cell Yield

BACKGROUND The success of an autologous fat graft depends in part on its total stromal vascular fraction (SVF) and adipose-derived stem cells (ASCs). However, variations in the yields of ASCs and SVF cells as a result of different harvesting techniques and donor sites are poorly understood. OBJECTIVE To investigate the effects of adipose tissue harvesting technique and donor site on the yield of ASCs and SVF cells. METHODS Subcutaneous fat tissues from the abdomen, flank, or axilla were harvested from patients of various ages by mechanical liposuction, direct surgical excision, or Colemans technique with or without centrifugation. Cells were isolated and then analyzed with flow cytometry to determine the yields of total SVF cells and ASCs (CD11b-, CD45-, CD34+, CD90+, D7-FIB+). Differences in ASC and total SVF yields were assessed with one-way analysis of variance. Differentiation experiments were performed to confirm the multilineage potential of cultured SVF cells. RESULTS Compared with Colemans technique without centrifugation, direct excision yielded significantly more ASCs (P < .001) and total SVF cells (P = .007); liposuction yielded significantly fewer ASCs (P < .001) and total SVF cells (P < .05); and Colemans technique with centrifugation yielded significantly more total SVF cells (P < .005), but not ASCs. The total number of SVF cells in fat harvested from the abdomen was significantly larger than the number in fat harvested from the flank or axilla (P < .05). Cultured SVF cells differentiated to adipocytes, osteocytes, and chondrocytes. CONCLUSIONS Adipose tissue harvested from the abdomen through direct excision or Colemans technique with centrifugation was found to yield the most SVF cells and ASCs.

Decellularized skin/adipose tissue flap matrix for engineering vascularized composite soft tissue flaps

UNLABELLED Using a perfusion decellularization protocol, we developed a decellularized skin/adipose tissue flap (DSAF) comprising extracellular matrix (ECM) and intact vasculature. Our DSAF had a dominant vascular pedicle, microcirculatory vascularity, and a sensory nerve network and retained three-dimensional (3D) nanofibrous structures well. DSAF, which was composed of collagen and laminin with well-preserved growth factors (e.g., vascular endothelial growth factor, basic fibroblast growth factor), was successfully repopulated with human adipose-derived stem cells (hASCs) and human umbilical vein endothelial cells (HUVECs), which integrated with DSAF and formed 3D aggregates and vessel-like structures in vitro. We used microsurgery techniques to re-anastomose the recellularized DSAF into nude rats. In vivo, the engineered flap construct underwent neovascularization and constructive remodeling, which was characterized by the predominant infiltration of M2 macrophages and significant adipose tissue formation at 3months postoperatively. Our results indicate that DSAF co-cultured with hASCs and HUVECs is a promising platform for vascularized soft tissue flap engineering. This platform is not limited by the flap size, as the entire construct can be immediately perfused by the recellularized vascular network following simple re-integration into the host using conventional microsurgical techniques. STATEMENT OF SIGNIFICANCE Significant soft tissue loss resulting from traumatic injury or tumor resection often requires surgical reconstruction using autologous soft tissue flaps. However, the limited availability of qualitative autologous flaps as well as the donor site morbidity significantly limits this approach. Engineered soft tissue flap grafts may offer a clinically relevant alternative to the autologous flap tissue. In this study, we engineered vascularized soft tissue free flap by using skin/adipose flap extracellular matrix scaffold (DSAF) in combination with multiple types of human cells. Following vascular reanastomosis in the recipient site, the engineered products successful regenerated large-scale fat tissue in vivo. This approach may provide a translatable platform for composite soft tissue free flap engineering for microsurgical reconstruction.

Anterolateral thigh adipofascial flap for correction of facial contour deformities and micromastia

The anterolateral thigh (ALT) flap has gained popularity, yet the donor site remains problematic. With increased knowledge of the vascular anatomy, we anticipated that we would be able to contour the ALT adipofascial flap when reconstructing facial deformities and micromastia without sacrificing skin at the donor site. A total of 24 cases of hemifacial atrophy and 1 case of micromastia underwent anterolateral thigh adipofascial flap transplantation with vascular anastomosis. All surgical reconstructions resulted in satisfactory results with minimal donor-site morbidity. The anterolateral thigh adipofascial perforator flap is an ideal choice for autologous tissue reconstruction with primary defatting.

Distally based dorsal pedal neurocutaneous flap for forefoot coverage.

The authors describe their experience with the use of the distally based dorsal pedal neurocutaneous flap for distal foot coverage. Ten patients underwent reconstruction with 13 flaps between 2004 and 2008. One patient suffered from a traffic accident and 9 from electrical injury. All of the soft tissue defects resulted in metatarsophalangeal joint and phalanx bone exposure. The size of the flaps ranged from 6 × 2 cm to 11 × 6 cm. The flaps were elevated based on intermediate or medial dorsal pedal nerves. Nine flaps were harvested in first stage to repair the distal foot. Among them, 3 showed partial necrosis in the distal region because of venous insufficiency. Four flaps underwent a surgical delay procedure in the first stage and were then transferred to reconstruct phalanx wounds in the second stage, surviving completely. All patients were satisfied with their reconstruction and donor site contour. The distally based dorsal pedal neurocutaneous flap can be used to repair the distal foot soft tissue defects, providing sufficient skin territory and excellent aesthetic and functional recovery. Surgical delay effectively enhances the distally based dorsal pedal neurocutaneous flap survival, particularly for the large size flaps.