Filip Stillaert

An arteriovenous loop in a protected space generates a permanent, highly vascular, tissue-engineered construct.

A major obstacle to 3‐dimensional tissue engineering is incorporation of a functional vascular supply to support the expanding new tissue. This is overcome in an in vivo intrinsic vascularization model where an arteriovenous loop (AVL) is placed in a noncollapsible space protected by a polycarbonate chamber. Vascular development and hypoxia were examined from 3 days to 112 days by vascular casting, morphometric, and morphological techniques to understand the models vascular growth and remodeling parameters for tissue engineering purposes. At 3 days a fibrin exudate surrounded the AVL, providing a scaffold to migrating inflammatory, endothelial, and mesenchymal cells. Capillaries formed between 3 and 7 days. Hypoxia and cell proliferation were maximal at 7 days, followed by a peak in percent vascular volume at 10 days (23.2±03.14% compared with 3.5±92.68% at 3 days, P<0.001). Maximal apoptosis was observed at 112 days. The protected space and spontaneous microcirculatory development in this model suggest it would be applicable for in vivo tissue engineering. A temporal window in a period of intense angiogenesis at 7 to 10 days is optimal for exogenous cell seeding and survival in the chamber, potentially enabling specific tissue outcomes to be achieved.—Lokmic, Z., Stillaert, F., Morrison, W. A., Thompson, E. W., Mitchell, G. M. An arteriovenous loop in a protected space generates a permanent, highly vascular, tissue‐engineered construct. FASEB J. 21, 511–522 (2007)

Human clinical experience with adipose precursor cells seeded on hyaluronic acid-based spongy scaffolds

Histioconductive approaches to soft-tissue defects use scaffolds seeded with lineage- and tissue-specific progenitors to generate tissue which should reside in equilibrium with adjacent tissue. Scaffolds guide histiogenesis by ensuring cell-cell and cell-matrix interactions. Hyaluronic acid-based (HA) preadipocyte-seeded scaffolds were evaluated for their adipo-conductive potential and efficacy in humans. Preadipocytes were isolated from lipoaspirate material and seeded on HA scaffolds. The cellular bio-hybrid (ADIPOGRAFT) and an acellular control scaffold (HYAFF11) were implanted subcutaneously. At specific time points (2, 8 and 16 weeks) explants were analyzed histopathologically with immunohistochemistry. No adverse tissue effects occurred. Volume loss and consistent degradation of the HYAFF11 scaffolds compared to the ADIPOGRAFT group indicated progressive tissue integration. No consistent histological differences between both groups were observed. By 8 weeks all void spaces within the scaffolds were filled with cells with pronounced matrix deposition in the ADIPOGRAFT bio-hybrids. Here we show that HA scaffolds were stable cell carriers and had the potential to generate volume-retaining tissue. However, no adipogenic differentiation was observed within the preadipocyte-seeded scaffolds.

A New Discriminative Criterion for the Development of Franz Diffusion Tests for Transdermal Pharmaceuticals

PURPOSE In vitro skin/membrane permeation profiling of topical pharmaceuticals is an important overall quality attribute in the evaluation of product consistency and it is also used for IVIVR (in vitro - in vivo relationship) purposes in product development and change control. Franz diffusion cell (FDC) experiments are emerging as a generally accepted methodology in this field, where the choice of operational conditions requires a data-supported justification towards the discriminating power of the test. A response function is therefore proposed to objectively quantify the discriminating power. METHODS We evaluated the usefulness of the proposed response function by studying one of the operational conditions, i.e. the influence of receptor medium composition, on the FDC in vitro penetration behaviour of the model compound testosterone formulated in four different topical preparations, using both artificial membranes and dermatomed human skin. RESULTS From the obtained cumulative amount of testosterone in the receptor fluid versus time curves, the permeability coefficient Kp of testosterone from each formulation was calculated. The evaluation of the discriminating power of the different media was performed using our new objective response function based upon an equal spread criterion of normalised Kp values. CONCLUSION We demonstrated significant differences in discriminating power between the different media used, with the overall best results obtained with hydroxypropyl-beta-cyclodextrine (HPBCD) containing media. The proposed new criterion was found to be useful for the rational design of an in vitro diffusion test for transdermal pharmaceuticals.

Complex facial reconstruction by vascularized composite allotransplantation: The first Belgian case

INTRODUCTION Complex injuries to the central part of the face are difficult to reconstruct with the current plastic surgery methods. The ultimate one-staged approach to restore anatomy and vital facial functions is to perform a vascularized composite allotransplantation (VCA). METHODS A 54-year-old man suffered from a high-energy ballistic injury, resulting in a large central facial defect. A temporary reconstruction was performed with a free plicated anterolateral thigh (ALT) flap. Considering the goal to optimally restore facial function and aesthetics, VCA was considered as an option for facial reconstruction. A multidisciplinary team approach, digital planning, and cadaver sessions preceded the transplantation. RESULTS A digitally planned facial VCA was performed involving the bilateral maxillae, the hard palate, a part of the left mandible, and the soft tissues of the lower two-thirds of the face. Due to meticulous preparations, minimal adjustments were necessary to achieve good fitting in the recipient. At week 17, a grade 4 rejection was successfully treated; sensory and motor recovery was noted to occur from the fourth postoperative month. Several serious infectious and medical problems have occurred until 15-months postoperatively; following that, the clinical situation has remained stable. Two years postoperatively, the patient and his family are very satisfied with the overall outcome and social reintegration in the community is successful. CONCLUSION The first face transplant in Belgium (#19 worldwide) was successful because of a meticulous 3-year preparation by a large multidisciplinary team. In our experience, preparatory cadaver dissections and three-dimensional (3D) computed tomographic (CT) modeling were valuable tools for an optimal intraoperative course and good alignment of the bony structures. Continuous long-term multidisciplinary follow-up is mandatory for surveillance of the complications associated with the immunosuppressive regime and for functional assessment of the graft.

The role of the internal mammary vessels as recipient vessels in secondary and tertiary head and neck reconstruction

BACKGROUND Successful microsurgical free tissue transfer for head and neck reconstruction highly depends on the quality of the recipient vessels. In most cases, vessels near the site of resection are available; however, when the bilateral vascular network in the neck is compromised or inaccessible due to prior surgery and/or irradiation, alternatives have to be sought. METHODS Secondary or tertiary head and neck reconstruction was performed using the internal mammary vessels (IMVs) as recipient vessels in seven patients who had undergone previous neck dissection and radiation therapy. Indications were: tracheal-oesophageal fistula or stenosis (n = 4), oesophageal-cutaneous fistula (n = 1), saliva fistula (n = 1) and oral cancer (n = 1). Free flaps used for reconstruction were radial forearm flap (FRFF) (n = 5), anterolateral thigh flap (ALT) (n = 3) and transverse rectus abdominis myocutaneous flap (TRAM) (n = 1). Within two patients an additional ALT flap was necessary for soft-tissue coverage and resurfacing of the neck. The IMVs were separately exposed in a standard fashion over the second or third rib. The pedicle of the flap was anastomosed anterograde and end-to-end to the recipient vessels in all cases. Mean pedicle length was 14.3 cm (11-20 cm), with a mean distance of 9.8 cm (7-13 cm) between the resection and recipient vessel site. RESULTS All patients were tumour free at time of re-operation and no sign of radiation injury was observed in the recipient vessels. All flaps survived and all patients healed without major complications. Mean follow-up time was 18 months. Four patients died of local recurrence or distant metastases during follow-up. CONCLUSION In the vessel-depleted neck, the IMVs are a reliable and easy accessible recipient area for microsurgical reconstruction of the head and neck. Surgical management and technique refinements for dissection of the vessels are discussed. In combination with free flaps with a long pedicle, especially perforator flaps, vein grafts are unnecessary and microsurgery can safely be performed outside the zone of injury.

Adipose tissue induction in vivo.

Engineering adipogenic tissue in vivo requires the concomitant induction of angiogenesis to generate a stable long-term three-dimensional construct. Histioconductive tissue engineering strategies have been used. The disadvantage of using biodegradable scaffolds is a delayed angiogenic induction resulting in ischemic necrosis of the central cell population in the scaffold. We evaluated an histioinductive approach for adipose tissue engineering by combining essential key components for adipogenic induction: (1) a precursor cell source, (2) a vascular pedicle, (3) a supportive matrix, and (4) a chamber to preserve space for the new tissue to develop. We observed concomitant adipogenic and angiogenic induction after 6 weeks in three-dimensional adipose tissue constructs.

Is a Second Free Flap Still an Option in a Failed Free Flap Breast Reconstruction

Background: Salvage of a failed autologous breast reconstruction is a complex and challenging problem. The purpose of this study was to analyze the indications, methods, and outcomes of tertiary surgery in patients with a failed autologous breast reconstruction. Methods: A retrospective chart review was performed for all patients who underwent breast reconstruction with autologous tissue performed by the senior author (M.H.) between 2002 and 2009. Special emphasis was made to evaluate the first reconstruction performed, causes of failure, indications for tertiary reconstruction, and outcomes. A preoperative hematologic workout was performed. For patients who were classified within the highest group of thromboembolism, specific prophylactic measurements were taken for the tertiary surgery. Results: Of 688 patients who underwent autologous breast reconstruction, a total of 14 patients required tertiary breast reconstruction. Hypercoagulability was found in three patients resulting from disorders such as lupus anticoagulant positivity and antiphospholipid syndrome. Six patients (43 percent) underwent a combination of local skin flaps and/or implant reconstructions. Eight patients (57 percent) underwent nine microvascular breast reconstructions: five superior gluteal artery perforator flaps, three transverse myocutaneous gracilis flaps, and one deep inferior epigastric artery perforator flap. Two of nine flaps (22 percent) required quaternary reconstructions because of a failure of the second free flap. Additional corrections such as revision lipofilling, scar revision, contralateral breast shaping, implant change, and capsulotomies were performed in 92.7 percent of the patients, with a mean follow-up of 37 months (range, 6 months to 7 years). Conclusions: Tertiary surgery after autologous breast reconstruction failure has limited options and further reoperations are often needed. Careful patient history and selective blood tests may reveal hidden coagulation disorders. When a second free flap is planned, primary and secondary antithrombotic therapy should be considered.

Intrinsics and dynamics of fat grafts: an in vitro study.

Background: Despite a revived interest in fat grafting procedures, clinicians still fail to demonstrate clearly the in vivo behavior of fat grafts as a dynamic tissue substitute. However, the basic principles in cellular biology teach us that cells can survive and develop, provided that a structural matrix exists that directs their behavior. The purpose of this in vitro study was to analyze that behavior of crude fat grafts, cultured on a three-dimensional laminin-rich matrix. Methods: Nonprocessed, human fat biopsy specimens (approximately 1 mm3) were inoculated on Matrigel-coated wells to which culture medium was added. The control group consisted of fat biopsy specimens embedded in medium alone. The cellular proliferation pattern was followed over 6 weeks. Additional cultures of primary generated cellular spheroids were performed and eventually subjected to adipogenic differentiation media. Results: A progressive outgrowth of fibroblast-like cells from the core fat biopsy specimen was observed in both groups. Within the Matrigel group, an interconnecting three-dimensional network of spindle-shaped cells was established. This new cell colony reproduced spheroids that functioned again as solitary sources of cellular proliferation. Addition of differentiation media resulted in lipid droplet deposition in the majority of generated cells, indicating the initial steps of adipogenic differentiation. Conclusions: The authors noticed that crude, nonprocessed fat biopsy specimens do have considerable potential for future tissue engineering-based applications, provided that the basic principles of developmental, cellular biology are respected. Spontaneous in vitro expansion of the stromal cells present in fat grafts within autologous and injectable matrices could create “off-the-shelf” therapies for reconstructive procedures.

Speech characteristics one year after first Belgian facial transplantation

Facial transplantation has progressed over the past 8 years. We did the first Belgian facial transplantation by vascularized composite tissue allotransplantation and report the 1‐year follow‐up regarding speech and oromyofunctional behavior.

Technical Recommendations for Penile Replantation Based on Lessons Learned from Penile Reconstruction

Penile amputation is an exceptional surgical emergency. Immediate replantation yields a high success and low complication rate. We report a case of a self-inflicted penile amputation treated with successful microsurgical replantation. Postoperative edema caused minor skin slough and temporary venous congestion was treated with medicinal leech therapy. Follow-up at 18 months showed normal subjective sensation; voiding and erectile function were present. Surgical management and technique refinements are discussed, based on a review of the literature and on our experience in penile reconstruction.