Engineered adipose tissue formation enhanced by basic fibroblast growth factor and a mechanically stable environment

Engineered adipose tissue can be used in plastic and reconstructive surgery to augment soft tissue lost due to mastectomy or lumpectomy. The three-dimensional space provided by a scaffold capable of withstanding in vivo compressive forces and neovascularization may promote engineered adipose tissue formation. The objective of this study was to determine whether voluminous adipose tissue can be engineered by combining a mechanically stable environment with basic fibroblast growth factor (bFGF). Mechanical support structures, fabricated from biodegradable synthetic polymers, were placed into subcutaneous pockets of athymic mice. Human preadipocytes, containing fibrin matrix, with (group 1) or without (group 2) bFGF were injected into the space created by the support structures. Additionally, human preadipocytes containing fibrin matrix, with (group 3) or without (group 4) bFGF, were injected into subcutaneous spaces without support structures. Six weeks after implantation, the original implant volume was approximately maintained in groups 1 and 2, whereas groups 3 and 4 showed significant implant shrinkage. Adipogenesis and angiogenesis were more extensive in the group 1 than any other group. The fraction of human nuclear antigen-positive adipocytes in the implant was highest in group 1. Mouse adipocyte-specific genes were also expressed in the implants, again at the highest levels in group 1. Implanted preadipocyte apoptosis was significantly reduced in the groups treated with bFGF (groups 1 and 3) as opposed to those without (groups 2 and 4). This study demonstrates that combining a mechanically stable environment with bFGF can promote voluminous adipose tissue regeneration. This adipogenesis was likely promoted by the mechanically stable three-dimensional space, enhanced neovascularization, implanted cell survival, and host adipogenic cell migration. The method described in this study could be useful to augment adipose tissue used in plastic and reconstructive surgery.     

Autologous in vivo adipose tissue engineering in hyaluronan-based gels--a pilot study

BACKGROUND: There is a major clinical need for strategies for adequately reconstructing the soft tissue defects found after deep burns, tumor resection, or trauma. A promising solution is adipose tissue engineering with preadipocytes, stem-cell derived precursors of the adipose tissue, implanted within biomaterials. This pilot study evaluated hyaluronan gels mixed with autologous undifferentiated preadipocytes in a pig model for their potency to generate new fat. MATERIALS AND METHODS: Preadipocytes were isolated from intra-abdominal pig fat by collagenase digestion, plated on fibronectin-coated culture dishes in Dulbecco's modified Eagle medium/Ham's F12 (Biochrom, Berlin, Germany) combined with 10% pig serum, expanded, and mixed with hyaluronan gel. Two types of gels with varying degrees of amidation of the carboxyl groups were tested (HYADD3, HYADD4). Cell-loaded gels and unseeded controls were injected subcutaneously into the ears of three pigs, explanted at 6 wk, and analyzed histologically. RESULTS: Both cell-loaded specimens were detected macroscopically. They demonstrated a slight volume effect with limited stability after 6 wk. Unloaded HYADD3 and HYADD4 controls could not be identified at the time of explantation. Histology of HYADD3 revealed islets of mature adipocytes and vessels embedded in fat tissue surrounded by gel. In contrast, no fat formation was found in HYADD4 gels when implanted in the ear. CONCLUSIONS: Histological findings demonstrate that HYADD3 is a promising gel for generating adipose tissue. Even though HYADD3 might be a potential material for the reconstruction of small tissue defects, the question remains as to whether the adipose tissue within the gel is attributable to preadipocyte maturation or ingrowth from neighboring tissue.     

Comparison of different fabrication techniques for human adipose tissue engineering in severe combined immunodeficient mice

Adipose tissue engineering has been advocated for soft‐tissue augmentation and for the treatment of soft tissue defects. The efficacy in terms of persistence of the engineered fat is, however, not yet understood and could depend on the nature of fabrication and application. The high metabolic demand of adipose tissue also points to the problem of vascularization. Endothelial cell (EC) cotransplantation could be a solution. Human adipose tissue‐derived stromal cells were seeded on collagen microcarriers and submitted to adipogenic differentiation (“microparticles”). In a first run of experiments, these microparticles were implanted under the skin of severe combined immunodeficient (SCID) mice (n = 45) with and without the addition of human umbilical vein ECs (HUVECs). A group of carriers without any cells served as control. In a second run, adipose tissue constructs were fabricated by embedding microparticles in fibrin matrix with and without the addition of HUVEC, and were also implanted in SCID mice (n = 30). The mice were sacrificed after 12 days, 4 weeks, and 4 months. Mature adipose tissue, fibrous tissue, and acellular regions were quantified on whole‐specimen histological sections. The implantation of microparticles showed a better sustainment of tissue volume and a higher degree of mature adipose tissue compared with adipose tissue constructs. Immunohistology proved obviously perfused human tissue‐engineered vessels. There was a limited but not significant advantage in EC cotransplantation after 4 weeks in terms of tissue volume. In groups with EC cotransplantation, there were significantly fewer acellular/necrotic areas after 4 weeks and 4 months. In conclusion, the size of the implanted tissue equivalents is a crucial parameter, affecting volume maintenance and the gain of mature adipose tissue. EC cotransplantation leads to functional stable vascular networks connecting in part to the host vasculature and contributing to tissue perfusion; however, the long‐term benefit depends on additional basic conditions that need further research.     

Bicompartmental Breast Lipostructuring

The techniques of additive mastoplasty described over the years require the use of alloplastic materials (silicon), which often are poorly tolerated by the body and need access paths that could leave visible, unaesthetic residual scars. Furthermore, the controversy over silicone gel-filled breast implants, which in the early 1990s restricted their clinical use for primary cosmetic breast augmentation, still raises concerns in some patients. The authors therefore felt encouraged to search for alternatives to breast implants and reconsider fat transfer. In fact, for almost a century, autologous adipose tissue has been used safely and with success in many other surgical fields for the correction of volumetric soft tissue defects. Its natural, soft consistency, the absence of rejection, and the versatility of use in many surgical techniques have always made autologous adipose tissue an ideal filling material. In the past, the authors used this technique, as originally described by Fournier (intraparenchymal, en bloc injection), for 41 patients. However, disappointed by a very high rate of complications and the almost complete reabsorption of the grafted fat, they quit using the procedure. An extensive literature review indicated that the complications observed were related only to technical errors and to the anatomic site of harvesting and implantation. The authors therefore developed a new method incorporating recent contributions in functional anatomy and fat transfer. Fat is harvested in a rigorously closed system, minimally manipulated, and reimplanted strictly in two planes only: into the retroglandular and prefascial space and into the superficial subcutaneous plane of the upper pole of the breast (bicompartmental grafting). Any intraparenchymal placement is carefully avoided. Since 1998, 181 patients (300 breasts) have undergone this procedure. Grafted fat volume has ranged from 160 to 685 ml (average, 325 ml) per breast. Complications have been minimal and temporary. All patients have been carefully monitored with preoperative and serial postoperative mammograms and ultrasonograms. This strict follow-up assessment allowed the authors to clarify the controversial aspect of microcalcifications, the main point of criticism for this procedure over the years. Microcalcifications can occur in response to any trauma or surgery of the breast, but are very different in appearance and location. Thus, they can be discriminated easily from those appearing in the context of a neoplastic focus. Probably the most important point is that the fat survival ranged from 40% to 70% at 1 year. The volume is maintained because when the authors transplant living fat tissue, they also transfer a consistent amount of adult mesenchymal stem cells that spontaneously differentiate into preadipocytes and then into adipocytes, compensating for the partial loss of mature adipocytes reabsorbed through time. This theory has been well demonstrated via advanced research performed by the authors and by many other prominent medical institutes worldwide. The findings show that adipose tissue has the same potential for growth of adult mesenchymal totipotential stem cells of bone marrow and can eventually be differentiated easily by the use of specific growing factors and according to the needs and applications in other cellular lines (osteogenic, chondrogenic, myogenic, epithelial). In summary, the authors wish to highlight a formerly controversial procedure that, thanks to recent technical and clinical progress, has become a safe and viable alternative to the use of alloplastic materials for breast augmentation for all cases in which additive mastoplasty with implants is either unsuitable or unacceptable by the patient herself. However this method cannot be considered yet as a complete substitute for augmentation with implants because the degree of augmentation and projection still is limited.     

Correction of breast contour deformities using polyurethane breast implant capsule in revisional breast surgery

Surgeons are commonly confronted with breast contour deformities and defects that result from previous surgical interventions. These soft tissue deformities can be corrected by conventional reconstructive flap surgery using autologous tissue, but there can be donor site morbidity. Smaller volume replacement is possible using temporary fillers such as hyaluronic acid or polylactic acid, or by using 'permanent' fillers such as autologous fat, but large defects are notoriously difficult to fill and often the fillers resorb or migrate. The patient described in this case report had an exchange of polyurethane implant (PU) in the left breast and correction of a contralateral breast contour filling deformity. A left breast partial capsulectomy was performed after implant removal and the capsule graft was inserted into a predissected pocket where soft tissue augmentation was required. A biopsy from the PU capsule was reported to show a foreign body type giant cell reaction to PU material in a fibrous capsule, lined by synovial metaplasia. The post-operative result showed satisfactory soft tissue revolumisation. PU breast implant structured capsule has thus been used as filler to correct breast soft tissue deformity and contour defects. Clearly it may have a use in other anatomical sites.     

Aspiration lipectomy and adipose tissue injection pathophysiologic commentary

Summary The subcutis, made up of connective and adipose tissue, may consist of one or two layers, depending on the part of the body. The superficial layer, up to 10 mm in thickness, has the function of giving contour. In localizations where one typically sees relatively large fat deposits a padding layer is added to the contour layer, and this may be centimeters in thickness. Aspiration lipectomy should be limited to reducing the padding layer. The advancing cannula perforates the supportive structures in the subcutis and breaks up the fat lobules. Analysis of the aspirated material suggests that residues of fat lobules remain adhering to the chamber walls; it may be assumed that they necrose and are removed by the mechanisms of foreign body reaction. Some stiffening of the reduced subcutis due to fibroproductive processes appears to be inevitable. Implantation of fat fragments obtained through lipectomy offers little biological advantage; at the same time one must expect extensive necrosis of the implanted tissue. With fat cell suspensions, failure to heal seems to be due to inadequate restoration of the capillary bed. The use of cultured preadipocytes to build up adipose tissue is an attractive idea, but the method is still at the experimental stage. Presented on the 19th Congress of the German Society for Plastic Surgery in Düsseldorf, September 27–30, 1989     

自体基质血管成分改善脂肪组织移植效果的实验研究

目的：分析自体脂肪基质血管成分（stromal vascular fraction cells,SVF）对脂肪颗粒（adipose granule,AG）移植的作用。方法：从6只健康新西兰家兔背部肩胛区获取脂肪组织,实验组将自体SVF与自体AG复合,植入家兔耳部皮下,对照为单纯脂肪移植。在术后1、3、6个月,用B超和游标卡尺测量移植脂肪体积;术后6个月取材常规组织学观察。结果：术后1、3、6个月对照组脂肪组织存活率分别为：（64.35±8.36）%、（58.22±2.88）%、（50.61±9.47）%;实验组脂肪组织存活率分别为：（77.42±5.1）%、（67.95±6.09）%、（72.75±4.37）%。两组比较均存在显著性差异（P〈0.05）。术后6个月组织学观察两组呈正常脂肪组织形态,未见明显差异。结论：自体SVF复合脂肪颗粒能够显著提高移植脂肪组织的成活率,为临床脂肪移植提供实验依据。     

Vascular augmentation of free adipose tissue grafts

Summary The effect of vascular implantation on the healing of free adipose tissue grafts was studied experimentally in rats. Adipose tissue resected from the parauterine pad was transplanted around the femoral vascular bundle. Two biological variants of the method were used: transplantation to (a) the freshly mobilized vascular bundle and (b) a bundle prepared three days previously. Implants made directly under the skin served as controls. Implantation around the freshly mobilized vascular bundle showed that healing of free adipose tissue implants is promoted by this technique: revascularization was augmented and the proportion of adipose tissue surviving was significantly greater than with the controls. With implantation into previously prepared tissue, there was evidence that healing may be further improved. The biological advantage thus gained was, however, largely impaired by the tissues being coated with fibrin; this prevented close contact between the graft and the recipient tissue.     

前脂肪细胞凝胶植入预防腰椎术后硬膜外腔瘢痕粘连的实验研究

目的：探讨自体前脂肪细胞凝胶植入法预防腰椎术后硬膜外腔瘢痕粘连的效果。方法：将60只SD大鼠随机分为A组(前脂肪细胞凝胶植入组)、B组(自体脂肪颗粒植入组)、C组(空白对照组)，将A组动物先进行前脂肪细胞培养，待其脂肪细胞培养传代至适量后，制备各组大鼠的椎板缺损模型，A组取培养的单层脂肪细胞与生物蛋白胶制成混合物，B组取自体脂肪颗粒分别植入椎板缺损区，C组不植入任何物质，于术后8周行大体、光镜、电镜及MRI检查。结果：A组预防硬膜外腔瘢痕粘连效果良好，优于B组。结论：自体前脂肪细胞与生物蛋白胶制成的混合物植入预防硬膜外腔瘢痕的形成与粘连有良好的效果，是一种预防椎板切除术后硬膜外腔瘢痕形成与粘连的新方法。     

A role for vacuum-assisted closure in lower limb trauma: a proposed algorithm

The British Association of Plastic Surgeons and British Orthopaedic Association estimate that 70% of Gustilo 3b tibial fractures require flap coverage. Prior to the introduction of vacuum-assisted closure (V.A.C.) in the late 1990s, options were limited for the treatment of such injuries in selected individuals with medical comorbidity or complex wounds. VAC has now become an important part of our algorithm for soft tissue reconstruction with excellent results, but its role has not yet been defined in common management pathways. The authors present 3 cases, all different in their mechanism of injury, type of injury, and subsequent management, but all had a successful outcome with VAC.     

脂肪组织长期储存的研究进展

自吸脂术出现以来,使用脂肪组织进行软组织填充已成为临床常见的治疗方法。但目前临床仍存在一处填充需要多次移植的情况。如能一次抽吸后对脂肪进行妥善保存,并在需要时进行再次注射,就可以减少抽脂次数,减轻患者痛苦。因此,对脂肪组织进行长期储存并减少其重吸收率具有重要的临床意义。本文就脂肪组织长期储存的研究进展进行综述。     

自体脂肪颗粒注射移植治疗面部软组织凹陷

目的 评价自体脂肪颗粒注射移植治疗面部软组织凹陷的效果。方法 利用吸脂术从身体其它部位皮下吸取脂肪颗粒，注射植入面部软组织凹陷部位。结果 1996年～2002年，共治疗36例(56个部位)面部软组织凹陷，其中45个部位接受1次脂肪颗粒注射，11个部位接受2次注射，每次注射量为1．5m1～24m1，平均8．9m1。在随访超过6个月的28例(43个部位)中，8个部位(18．6％)注射2次，术后6个月38个部位外形得到明显隆起的改善效果，优良率88．4％(38／43)。未见严重并发症。结论 对于较为单纯的面部软组织凹陷的患，自体脂肪颗粒注射移植是一种微创、简便、安全、有效的治疗方法，必要时需重复注射。     

Clinical and instrumental evaluation of implant stability after free fibula flaps for jaw reconstruction

Osseointegration is a direct union between bone and an allograft with no soft tissue interposition. It represents the morphological basis of secondary stability. Introduction of Resonance Frequency Analysis (RFA) as a commercially available technique has made it possible to measure implant stability as an Implant Stability Quotient (ISQ) unit at any time during the course of implant treatment and loading. The study aims to clinically and objectively analyze the stability of implants loaded upon revascularized free fibula flaps on patients who underwent maxilla or mandible reconstruction following trauma, oncological resection or class V–VI Cawood's atrophy treatment. Of the 105 patients that underwent jaw bone reconstruction with free fibula flaps in our division between 1993 and 2003, 37 were eligible for implant-supported dental rehabilitation. Of these, the last 12 were selected and included in the study. Primary and secondary implant stability assessments involved a clinical evaluation, radiograms and RFA measurements using the Osstell (Integration Diagnostics, Savedalen, Sweden) at the time of implant insertion, at 6 months and at 1 year of functional loading. 76 implants were loaded in 12 patients. All of them were clinically stable at each evaluation. Radiograms showed modest bone resorption after 1 year of functional load in 16 implants (21%). Implant stability levels ranged from 48 to 74 ISQ (mean 59.9±7.8 ISQ) at 6 months. At 1 year, the mean ISQ level was higher (62.6±7.2) but still coherent according to the radiographic evidence. Implant-supported dental rehabilitation of the reconstructed jawbone is not always possible. Free fibula flap represents a good substrate for implant osseointegration being a bicortical bone capable of structural remodelling after masticatory load enhancement. When possible, instrumental evaluation of implant stability with RFA and supported by radiograms represent the best objective way to assess osseointegration as a clinical evidence alone is unable to offer a definitive assessment.Presented at the 16th Annual Meeting of the European Association of Plastic Surgeons (EURAPS), May 26–28, 2005, Marseille, France.     

Injectable gellan gum hydrogels with autologous cells for the treatment of rabbit articular cartilage defects

In this work, the ability of gellan gum hydrogels coupled with autologous cells to regenerate rabbit full‐thickness articular cartilage defects was tested. Five study groups were defined: (a) gellan gum with encapsulated chondrogenic predifferentiated rabbit adipose stem cells (ASC + GF); (b) gellan gum with encapsulated nonchondrogenic predifferentiated rabbit adipose stem cells (ASC); (c) gellan gum with encapsulated rabbit articular chondrocytes (AC) (standard control); (d) gellan gum alone (control); (e) empty defect (control). Full‐thickness articular cartilage defects were created and the gellan gum constructs were injected and left for 8 weeks. The macroscopic aspect of the explants showed a progressive increase of similarity with the lateral native cartilage, stable integration at the defect site, more pronouncedly in the cell‐loaded constructs. Tissue scoring showed that ASC + GF exhibited the best results regarding tissue quality progression. Alcian blue retrieved similar results with a better outcome for the cell‐loaded constructs. Regarding real‐time PCR analyses, ASC + GF had the best progression with an upregulation of collagen type II and aggrecan, and a downregulation of collagen type I. Gellan gum hydrogels combined with autologous cells constitute a promising approach for the treatment of articular cartilage defects, and adipose derived cells may constitute a valid alternative to currently used articular chondrocytes. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1193–1199, 2010     

The management of compound leg injuries in the West Midlands (UK): are we meeting current guidelines?

The joint British Association of Plastic Surgeons and British Orthopaedic Association (BAPS/BOA) guidelines for the management of patients with open tibia fractures were published in 1991 and 1997 and provided a synopsis of injury epidemiology and best care. We present a retrospective review of 66 patients treated over a 2-year period (January 2001-February 2003) at a regional plastic surgery unit. There were 33 direct admissions and 33 transfers from other hospitals in which the average delay in transfer was 7.8 days (1-28) and in whom 26 (79%) patients had already undergone surgery. Plastic surgeons were involved in 46 (62%) of the 66 patient cohort and 27 (82%) transferred patients. The delay after admission until soft tissue cover was 3.8 days (0-15). Twenty-nine (44%) complications were recorded, 20 (69%) of these were in the transferred group and additional orthopaedic intervention was needed in 11 (42%) of patients who had been operated on in other hospitals. Despite widespread dissemination and teaching of the BAPS/BOA guidelines, complex extremity trauma is often not managed well in our region. There are unacceptable delays in admission, late communication, poor note keeping and follow up. The initial surgery/fixation is often sub-optimal and soft tissue reconstruction has to be considered at the least advantageous time period for the patient, leading to an increased hospital stay and complications.     

Clinical and Experimental Study of Autologous Fat Grafting After Processing by Centrifugation and Serum Lavage

Background  This clinical and experimental study compared adipose tissue transplant behavior after two different techniques of purifying: centrifugation at 3400 rpm for 3 min and serum lavage without centrifugation. Methods  Clinical evaluation was performed under standardized conditions for lipofilling on a series of 51 female patients, intentionally selected to have similar characteristics and assigned to two groups based on the method of processing. Experimentally, a culture system in diffusion chambers with vitaline membranes was designed to mimic the behavior and to study the morphology of the adipose tissue used for autografting. Survival, structure, and proliferation of the adipose cells in vitro were examined by classical histologic H&E staining and immunohistochemistry for leptin and cyclin D1. Results  The main differences encountered experimentally were the presence of a greater amount of preadipocytes in the noncentrifuged adipose tissue cultures and more distinctly expressed cell proliferation. The postoperative clinical results favored of the serum lavage purifying technique. Conclusion  Our data suggest that with transplantation of noncentrifuged adipose tissue more active preadipocytes are applied which could possibly lead to better potential chances of survival and even de novo development of fat.     

Hydroxyapatite orbital implant covered with fascia lata in post-enucleation eye reconstruction

The loss of an eye is a highly traumatic event, with severe psychological and physical implications. From November 1996 to June 2002, 32 patients underwent eye enucleation and placement of a hydroxyapatite orbital implant wrapped in autologous fascia lata; this was then fixed to the four rectus muscles. Motility was noted in all reconstructed sockets, without implant related complications. The immediate post-operative period was uncomplicated and all patients were discharged within 48 to 72 h after surgery. The orbital implant remained well positioned without dehiscence and motility was satisfactory. The follow-up ranged from 8 months to more than 5 years.Presented at the 14th Annual Meeting of the European Association of Plastic Surgeons (EURAPS), Vienna, Austria, 29–31 May 2003     

Transcolumellar incision for correction of unilateral cleft lip nose

Summary The best way I have found to correct the adult cleft lip nose with marked deformity is to expose the cartilaginous framework and re-align it in improved position. The anterior transcolumellar incision combined with the vestibular extensions is effective. It is important that the cleft side alar cartilage be shifted to the desired position, without any tension in the lateral vestibule, such as frequently results from an oro-nasal fistula. If the cartilaginous framework is correctly realigned, the manipulation of the soft tissue at the anterior nostril margin by elliptical excision or rolling the marginal skin into the vestibule to create the new margin is not necessary in the great majority of cases.Presented in part at the Annual Meeting of the Japanese Association of Plastic Surgeons on April 6, 1979 in Tokyo     

Tissue engineering of human fat for soft tissue augmentation: in vitro results

. In our study we investigated whether human preadipocytes from human fat derived from routine abdominoplasty were suitable for the in vitro cultivation of fat. We could isolate and culture preadipocytes from patients of different ages and stimulate the cultured cells to differentiate into adipocytes and accumulate fat. We noticed age-related differences in the growth rate and differentiation capability into mature adipocytes. We believe that in the future a cell suspension of preadipocytes from a patient amplified in tissue culture might provide a relatively unlimited amount of injectable autologous transplant to fill soft tissue defects.