Robert T. Grant

tissue Engineered Bone Repair of Calvarial Defects Using Cultured Periosteal Cells

&NA; Periosteum has been demonstrated to have cell populations, including chondroprogenitor and osteoprogenitor cells, that can form both cartilage and bone under appropriate conditions. In the present study, periosteum was harvested, expanded in cell culture, and used to repair critical size calvarial defects in a rabbit model. Periosteum was isolated from New Zealand White rabbits, grown in cell culture, labeled with the thymidine analog bromodeoxyuridine for later localization, and seeded into resorbable polyglycolic acid scaffold matrices. Thirty adult New Zealand White rabbits were divided into groups, and a single 15‐mm diameter full‐thickness calvarial defect was made in each animal. In group I, defects were repaired using resorbable polyglycolic acid implants seeded with periosteal cells. In group II, defects were repaired using untreated polyglycolic acid implants. In group III, the defects were left unrepaired. Rabbits were killed at 4 and 12 weeks postoperatively. Defect sites were then studied histologically, biochemically, and radiographically. In vitro analysis of the cultured periosteal cells indicated an osteoblastic phenotype, with production of osteocalcin upon 1,25(OH)2 vitamin D3 induction. In vivo results at I weeks showed islands of bone in the defects repaired with polyglycolic acid implants with periosteal cells (group I), whereas the defects repaired with untreated polyglycolic acid implants (group II) were filled with fibrous tissue. Collagen content was significantly increased in group I compared with group II (2.90 ± 0.80 &mgr;g/mg dry weight versus 0.08 ± 0.11 &mgr;g/mg dry weight, p < 0.006), as was the ash weight (0.58 ± 0.11 mg/mg dry weight versus 0.35 ± 0.06 mg/mg dry weight, p < 0.015). At 12 weeks there were large amounts of bone in group I, whereas there were scattered islands of bone in groups II and III. Radiodensitometry demonstrated significantly increased radiodensity of the defect sites in group I, compared with groups II and III (0.740 ± 0.250 OD/mm2 versus 0.404 ± 0.100 OD/mm2 and 0.266 ± 0.150 OD/mm2, respectively, p < 0.05). Bromodeoxyuridine label, as detected by immunofluorescence, was identified in the newly formed bone in group I at both 4 and 12 weeks, confirming the contribution of the cultured periosteal cells to this bone formation. This study thus demonstrates a tissue‐engineering approach to the repair of bone defects, which may have clinical applications in craniofacial and orthopedic surgery.

Gene-enhanced tissue engineering: applications for bone healing using cultured periosteal cells transduced retrovirally with the BMP-7 gene.

Periosteum has cell populations, including osteoprogenitor and chondroprogenitor cells, that can be grown in cell culture and form both bone and cartilage under appropriate conditions. The authors have shown previously that cultured periosteal cells can be used in the tissue engineering of bone, and they demonstrated substantial bone formation in a rabbit cranial defect model. In the current study, principles of tissue engineering were combined with principles of gene therapy to produce cultured periosteal cells transduced retrovirally with the bone morphogenetic protein 7 (BMP-7) gene to be used in the treatment of bone defects. Human BMP-7 complementary deoxyribonucleic acid was generated from a cell line using reverse transcription polymerase chain reaction and cloned into a retroviral vector plasmid. Retroviral vector particles were then used to transduce New Zealand White rabbit periosteal cells. Transduced periosteal cells demonstrated substantial production of both BMP-7 messenger ribonucleic acid by Northern blot analysis and BMP-7 protein by enzyme-linked immunosorbent assay. These cells were then seeded into polyglycolic acid (PGA) matrices and used to repair critical-size rabbit cranial defects. At 12 weeks, defect sites repaired with BMP-7-transduced periosteal cells/PGA had significantly increased radiographic and histological evidence of bone repair compared with those defect sites repaired with negative control-transduced cells/PGA, nontransduced cells/PGA, PGA alone, or unrepaired defects. Thus, this study demonstrates successfully a tissue engineering approach to bone repair using genetically modified cells.

Gene-enhanced tissue engineering : Applications for wound healing using cultured dermal fibroblasts transduced retrovirally with the PDGF-B gene

The treatment of difficult wounds remains a considerable clinical challenge. The goal of this study was to determine whether genetic augmentation of dermal cells on resorbable matrices can stimulate the healing process, leading to increased tissue repair in a rat full-thickness excisional wound repair model. The human platelet-derived growth factor B (PDGF-B) gene was the initial gene chosen to test this hypothesis. The human PDGF-B gene was obtained from human umbilical vein endothelial cells (HUVEC) by reverse transcriptase-polymerase chain reaction, cloned into retroviral vectors under control of either the cytomegalovirus promoter or the rat beta-actin promoter, and introduced into primary rat dermal cells. In vitro results demonstrate that rat dermal cells are transduced and selected readily using retroviral vectors, and engineered to secrete PDGF-B at a steady-state level of approximately 2 ng per milliliter culture per 1 million cells per 24 hours. Seeding of the gene-modified cells onto polyglycolic acid (PGA) scaffold matrices and introduction into the rat model resulted in substantially increased fibroblast hypercellularity over control wounds at both 7 and 14 days posttreatment. Our results demonstrate that gene augmentation of rat dermal fibroblasts with the PDGF-B gene introduced into this animal model via PGA matrices modulates wound healing and suggests that experimentation with additional genes for use separately or in combination with PDGF-B for additional, improved wound healing is warranted.

Lentiviral transfection with the PDGF-B gene improves diabetic wound healing.

Background: The treatment of diabetic wounds remains a difficult challenge. The present study investigates whether platelet-derived growth factor (PDGF) lentiviral gene therapy can improve diabetic wound healing in the diabetic db/dbmouse. Methods: PDGF cDNA was cloned and lentiviral vectors were constructed with either the PDGF-B or green fluorescence protein (GFP) gene. A 2 × 2-cm full-thickness dermal wound was made on each db/db mouse. Animals were divided into three groups, with eight animals in each group as follows: group I, empty wound; group II, lentiviral PDGF; and group III, lentiviral GFP. Lentiviral vectors were injected into the wounds and healing was assessed at 21 days. Harvested wounds were assessed for residual epithelial gap, granulation tissue area, PDGF expression, collagen formation (picrosirius red), and angiogenesis (CD31 staining). Results: Lentiviral vectors were constructed and transfected dermal fibroblasts demonstrated in vitro production of PDGF mRNA as measured by reverse-transcriptase polymerase chain reaction. Immunohistochemistry for PDGF confirmed successful in vivo transfection of the PDGF gene. At 21 days, reepithelialization and granulation tissue area were similar in all groups. However, there was a statistically significant increase in angiogenesis and substantially thicker, more coherently aligned collagen fibers in the PDGF group compared with controls. Conclusions: PDGF lentiviral vectors were successfully transfected into the regenerated dermis in diabetic wounds. Although reepithelialization was similar among the groups, there was enhanced angiogenesis and collagen deposition in the lentiviral PDGF group. These results demonstrate that lentiviral PDGF transfection of the diabetic wound enhances PDGF production, improves vascularization and collagen organization, and has potential clinical applications in diabetic wound treatment.

Use of topical sRAGE in diabetic wounds increases neovascularization and granulation tissue formation.

Advanced glycation end products (AGEs) accumulate in diabetic wounds as a result of the glycosylation of various proteins. Interaction of AGEs with the receptor for AGEs (RAGE) results in an exaggerated inflammatory response and compromised collagen production. These changes lead to impaired wound healing. A soluble form of RAGE (sRAGE) has been shown to bind AGEs and thereby blunt their pathogenetic effects. Using genetically diabetic C57BLks-db/db mice, the authors applied sRAGE topically to standardized full-thickness wounds to improve diabetic wound healing. They measured various parameters of wound healing such as neovascularization, reepithelialization, collagen formation, and granulation tissue area. Their results showed a statistically significant increase in granulation tissue area and microvascular density in the sRAGE group compared with untreated wounds. There was a trend toward a smaller epithelial gap in the sRAGE-treated group that did not reach statistical significance. The authors conclude that sRAGE may be a powerful treatment of accelerating diabetic wound healing.

Accelerated diabetic wound healing using cultured dermal fibroblasts retrovirally transduced with the platelet-derived growth factor B gene.

The treatment of diabetic wounds is a considerable clinical challenge. In this study, mouse dermal fibroblasts retrovirally transduced with the human platelet-derived growth factor B (PDGF-B) gene were used to treat diabetic mouse wounds. The PDGF-B gene was obtained from human umbilical vein endothelial cells, cloned into retroviral vectors, and introduced into diabetic mouse C57B1/ks-db/db dermal fibroblasts. In vitro results demonstrated production of PDGF-B protein by these transduced cells at steady-state levels of 1000 ng PDGF-B/106 cells/24 hours, and expression of PDGF-B mRNA. These cells were seeded onto polyglycolic acid scaffold matrices and used to treat diabetic mouse 20-mm × 20-mm full-thickness excisional dorsal skin wounds. Measurement of the residual epithelial gap at 21 days showed significantly accelerated healing (P < 0.05) of wounds treated with PDGF-transduced cells (epithelial gap 10.46 ± 1.20 mm) compared with untreated wounds (14.66 ± 0.591 mm), wounds treated with polyglycolic acid alone (14.80 ± 0.575 mm), or wounds treated with negative control LNCX-transduced cells (13.76 ± 0.831 mm). Immunohistochemical staining showed intense staining for PDGF in wounds treated with PDGF-B–transduced cells. This study demonstrates the promising potential for gene therapy in diabetic wound healing.

The absorbable dermal staple device: a faster, more cost-effective method for incisional closure.

Background: Closure with dermal sutures is time consuming, may increase the risks of inflammation and infection secondary to foreign body reaction, exposes the surgeon to possible needlestick injuries, and has variable cosmetic outcomes depending on each surgeons technique. The absorbable INSORB dermal stapler is hypothesized to be faster and more cost effective than sutures for dermal layer closures and provides a safer and more consistent result. Methods: This is a prospective, randomized, controlled study. Patients undergoing bilateral breast reconstruction with tissue expanders had one incision randomized to dermal closure with absorbable dermal staples. The contralateral side was closed with dermal sutures. During the expansion period, wounds were assessed by a blinded plastic surgeon using the 13-point Vancouver Scar Scale. At the time of implant exchange, both scars were excised and examined for histologic signs of inflammation. Results: Eleven patients (22 incisions) were enrolled in the study. The dermal stapler was four times faster than standard suture closure, reducing closure time by 10.5 minutes (p ≤ 0.001). Overall cost savings with the dermal stapler was

Objective Comparison of Commercially Available Breast Implant Devices

220 per case. In the early postoperative period, the dermal stapler had a higher Vancouver Scar Scale score than sutures because of superior wound eversion, a beneficial characteristic for wound healing. By 4 months postoperatively, no significant difference in scar scores was found between interventions. At 6 months, histologic analysis suggested decreased inflammatory cell invasion of the dermal stapler-closed scar. Conclusion: Closure using the absorbable dermal stapler can be performed significantly faster than standard suture closure techniques, allowing for a more cost-effective incisional closure with equivalent cosmetic results.

Clear to Cloudy: Silicone Breast Implants In Vivo

Breast implants are frequently used for both cosmetic breast augmentation and breast reconstruction after mastectomy. Three companies currently offer FDA-approved breast implants (Allergan, Mentor, and Sientra), but their product offerings—including permanent breast implants, breast tissue expanders, sizers, and post-operative warranty—can be difficult to compare because of brand names and company-specific jargon. The ability to have a brand-agnostic understanding of all available options is important for both the surgical trainee as well as the surgeon in clinical practice. After a brief review of the history of breast implant devices, this review utilizes a unique conceptual framework based on variables such as fill material, shape, relative dimensions, and surface coating to facilitate a better understanding of the similarities and differences between the different company’s offerings. Specifically, we identify which types of devices are offered by all three companies, those that are offered by only one company, those that have very limited product offerings, and those combinations that are not available at all. Finally, clinical implications are drawn from this framework that can be used by both cosmetic and reconstructive surgeons to counsel patients about all available options. Importantly, this project is entirely independent of any company’s funding, support, or input.Level of Evidence VThis journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Expanding the Criteria for Nipple-Sparing Mastectomy in Patients With Poor Prognostic Features

Level of Evidence V This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of