John N. Jensen

Increased free fat-graft survival with the long-term, local delivery of insulin, insulin-like growth factor-I, and basic fibroblast growth factor by PLGA/PEG microspheres.

&NA; The present investigation evaluates the effects of longterm, local delivery of insulin, insulin‐like growth factor‐1 (IGF‐1), and basic fibroblast growth factor (bFGF) on fatgraft survival using a poly (lactic‐co‐glycolic‐acid)‐polyethylene glycol (PLGA/PEG) microsphere delivery system. Twelve‐micrometer PLGA/PEG microspheres incorporated separately with insulin, IGF‐1, and bFGF were manufactured using a double‐emulsion solvent‐extraction technique. Inguinal fat from Sprague Dawley rats was harvested, diced, washed, and mixed with (1) insulin microspheres, (2) insulin‐like growth factor‐1 microspheres, (3) basic fibroblast growth factor microspheres, (4) a combination of the insulin and IGF‐1 microspheres, and (5) a combination of insulin, IGF‐1, and bFGF microspheres. The treated fat grafts were implanted autologously into subdermal pockets in six animals for each group. Animals receiving untreated fat grafts and fat grafts treated with blank microspheres constituted two external control groups (six animals per external control group). At 12 weeks, all fat‐graft groups were compared on the basis of weight maintenance and a histomorphometric analysis of adipocyte area percentage, indices of volume retention and cell composition, respectively. Weight maintenance was defined as the final graft weight as a percent of the implanted graft weight. All growth factor treatments significantly increased fat‐graft weight maintenance objectively, and volume maintenance grossly, in comparison with the untreated and blank microsphere‐treated controls. Treatment with insulin and IGF‐1, alone or in combination, was found to increase the adipocyte area percentage in comparison with fat grafts treated with bFGF alone or in combination with other growth factors. In conclusion, the findings of this study indicate that long‐term, local delivery of growth factors with PLGA/PEG microspheres has the potential to increase fat‐graft survival rates. Further, the type of growth factor delivered may influence the cellular/stromal composition of the grafted tissue. (Plast. Reconstr. Surg. 105: 1712, 2000.)

De novo adipose tissue generation through long-term, local delivery of insulin and insulin-like growth factor-1 by PLGA/PEG microspheres in an in vivo rat model: A novel concept and capability

&NA; This study was undertaken to characterize the duration of long‐term growth factor delivery by poly(lactic‐co‐glycolicacid)‐polyethylene glycol (PLGA/PEG) microspheres and to evaluate the potential of long‐term delivery of insulin and insulin‐like growth factor‐1 (IGF‐1) for the de novo generation of adipose tissue in vivo. PLGA/PEG microspheres containing insulin and IGF‐1, separately, were produced by a double‐emulsion solvent‐extraction technique. In the first phase of the experiment, the in vitro release kinetics of the microspheres were evaluated for the optical density and polyacrylamide gel electrophoresis of solutions incubated with insulin‐containing microspheres for four different periods of time (n = 1). The finding of increased concentrations of soluble insulin with increased incubation time confirmed continual protein release. In the second stage of the experiment, 16 rats were divided equally into four study groups (insulin, IGF‐1, insulin + IGF‐1, and blank microspheres) (n = 4). Insulin and IGF‐1 containing microspheres were administered directly to the deep muscular fascia of the rat abdominal wall to evaluate the potential for de novo adipose tissue generation via adipogenic differentiation from native nonadipocyte cell pools in vivo. Animals treated with blank microspheres served as an external control group. At the 4‐week harvest period, multiple ectopic islands of adipose tissue were observed on the abdominal wall of the animals treated with insulin, IGF‐1, and insulin + IGF‐1 microspheres. Such islands were not seen in the blank micro sphere group. Hematoxylin and eosin‐stained sections of the growth factor groups demonstrated mature adipocytes interspersed with fibrous tissue superficial to the abdominal wall musculature and continuous with the fascia. Oil‐Red‐O stained sections demonstrated that these cells contained lipid. Computer‐aided image analysis of histologic sections confirmed that there were statistically significant increases in the amount of “ectopic” adipose neotissue developed on the abdominal wall of animals treated with growth factor microspheres. In conclusion, this study confirms the long‐term release of proteins from PLGA/PEG microspheres up to 4 weeks and demonstrates the potential of long‐term local insulin and IGF‐1 to induce adipogenic differentiation to mature lipid‐containing adipocytes from nonadipocyte cell pools in vivo at 4 weeks. (Plast. Reconstr. Surg. 105: 1721, 2000.)

The spectrum of complications of immunosuppression: Is the time right for hand transplantation?

• Life-threatening complications of long-term immunosuppression include malignancy, infection, and metabolic disorders such as renal failure and diabetes. • Up to three-fourths or more of patients on chronic immunosuppressive medications experience an infectious complication. • The hand transplants to date have had multiple episodes of acute rejection. • The frequency and timing of episodes of acute rejection, even if the episodes are easily treated, are predictive of chronic allograft dysfunction and failure. • Chronic allograft rejection is not effectively treated with current immunosuppressive medications, and it has become a primary cause of long-term allograft failure. The introduction of cyclosporine A in the early 1980s revolutionized solid organ transplantation. There were marked improvements not only in preserving graft function, but also in prolonging patient survival following solid organ transplantation 1. As the viability of transplanted organs improved, the indications for clinical transplantation expanded to include not only acutely life-threatening conditions, but also more chronic conditions that adversely affected longevity and quality of life. Composite tissue allotransplantation involves the transplantation of nonvital tissues for reconstruction of deficits following trauma or tumor resection. If successful, composite tissue allotransplantation would facilitate the recovery of lost function through grafting of complex, disparate tissue types. However, composite tissue allotransplantation also poses unique challenges. Solid organs, such as the kidneys, may be more tolerant of rejection episodes than are composite tissues, for which the risk of rejection of one or more components is high. Parenchymal organs may have a tremendous functional reserve so that a substantial amount of tissue can be lost before organ function is compromised, but composite tissue allografts tend to have complex architecture with limited built-in redundancy. Thus, while the majority of kidney function may be lost before blood urea nitrogen or creatinine levels are elevated, comparable loss of the function of composite …

FK506 Rescues Peripheral Nerve Allografts in Acute Rejection

This study investigated the ability of the immunosuppressant FK506 to reverse nerve allograft rejection in progress. Eighty-four Buffalo rats received posterior tibial nerve grafts from either Lewis or Buffalo donor animals. Allografts were left untreated for either 7, 10, or 14 days before receiving daily subcutaneous FK506 injections (2 mg/kg). Time-matched control animals received either an isograft, an allograft with continuous FK506, or an allograft with no postoperative FK506 therapy. All animals underwent weekly evaluation of nerve function by walking track analysis. Experimental group animals were sacrificed either immediately prior to initiation of FK506 therapy (days 7, 10, or 14), after 2 weeks of immunosuppressive treatment, or 8 weeks postsurgery. Histomorphometric analysis, consisting of measurements of total number of nerve fibers, neural density, and percent of neural debris, demonstrated a statistically significant increase in regeneration in the isograft group relative to the untreated allograft group within 28 days of transplantation. Grafts harvested from animals receiving 2 weeks of FK506 after 7 or 10 days of rejection were histomorphometrically similar to time-matched isografts. By contrast, grafts from animals receiving 2 weeks of FK506 following 14 days without therapy resembled untreated allografts and demonstrated significant histomorphometric differences from isografts at the corresponding time point. Analysis of walking track data confirmed that relative to untreated allografts, functional recovery was hastened in animals receiving an isograft, or allograft treated with FK506. This study demonstrated that when started within 10 days of graft placement, FK506 could reverse nerve allograft rejection in rats evaluated following 2 weeks of treatment.

Antibody to transforming growth factor beta reduces collagen production in injured peripheral nerve

&NA; Epineurial scarring in peripheral nerve after injury inhibits normal axonal regeneration primarily due to fibroblast deposition of type I collagen. The transforming growth factor beta (TGF‐&bgr;) family is an important class of signaling molecules that has been shown to stimulate fibroblasts to produce collagen. The aim of this study was to design a prototypic therapeutic system in which the neutralization of TGF‐&bgr; in crushed rat sciatic nerve would decrease collagen formation. A total of 45 experimental Lewis rats were used. Group 1 animals (20 rats) sustained a unilateral crush injury to the sciatic nerve with injection of phosphate buffer solution. Group 2 animals (20 rats) sustained a unilateral crush injury to the sciatic nerve with injection of phosphate‐buffered saline and goat, anti‐rat, panspecific TGF‐&bgr; antibody. Group 3 control animals (five rats) underwent only exposure of sciatic nerve with injection of antibody. All animals were killed at 14 days and sciatic nerve specimens were harvested at that time. Slides of experimental tissue were processed using a 35S‐labeled oligomer for procollagen alpha‐1 mRNA, then dipped in photographic emulsion and examined by darkfield autoradiography. Morphometric analysis of pixel counts was then performed. A significant reduction in total pixel count per high‐power field and in total number of fibroblasts per high‐power field was found in crushed rat sciatic nerve treated with anti‐TGF‐&bgr; antibody when compared with those treated only with phosphate‐buffered saline. These findings are consistent with successful reduction in procollagen induction after a crush injury by topical administration of blocking antibody against transforming growth factor beta. The concept of growth factor blockade for therapeutic collagen reduction is attractive in the context of nerve injury, and the current article provides a model for future clinical application. (Plast. Reconstr. Surg. 102: 1100, 1998.)

Effect of FK506 on peripheral nerve regeneration through long grafts in inbred swine

Numerous small-animal studies have demonstrated that FK506 enhances nerve regeneration and accelerates functional recovery after nerve injury. However, no experimental study has corroborated these neuroregenerative effects in larger animals. This study investigated the effects of FK506 on nerve regeneration in inbred miniature swine. Eight animals received 8-cm ulnar nerve autografts and allografts. Treated animals received 0.1 to 0.4 mg/kg FK506 injections twice weekly to maintain immunosuppressive serum FK506 levels. At 24 weeks posttransplant, nerve grafts were harvested for histomorphometric analysis. Mixed lymphocyte cultures demonstrated alloreactivity in 1 treated animal and all untreated animals. In autografts, mean fiber count, nerve density, and percent neural tissue were doubled with FK506 therapy. In allografts, significant neuroregeneration was observed in animals treated with FK506, whereas untreated animals had no regeneration. Treatment with FK506 resulted in a trend toward enhanced axonal regeneration through nerve autografts and allografts in a large-animal model with defined histocompatibility barriers.

Augmentation of adipofascial flaps using the long-term local delivery of insulin and insulin-like growth factor-1.

The adipofascial flaps currently described in the literature frequently lack the volume requirements for reconstructive goals. In this study, the authors examined the use of long-term local delivery of insulin and insulin-like growth factor-1 (IGF-1) using polylactic-coglycolic acid/polyethylene glycol (PLGA/PEG) microspheres to augment inguinal adipofascial flaps based on the inferior epigastric vessels in the rat. Two flap models, the island flap and the limited dissection flap, were used to demonstrate simultaneous treatment and pretreatment modalities, respectively. Experimental groups received 12.5 mg of insulin microspheres (carrying 1 IU of insulin) plus 12.5 mg of IGF-1 microspheres (carrying 2.5 &mgr;g of IGF-1). A group undergoing the operation only (no treatment with microspheres) and a group treated with blank microspheres (no growth factor) served as external controls for the surgical procedure and the drug delivery device, respectively. In all groups (n = 5 animals in each), the contralateral flap served as an internal control. Upon harvest on postoperative day 28, the insulin and IGF-1–treated flaps in both models weighed statistically more than the internal control flaps and the two external control flaps. Likewise, on gross inspection, the adipogenic growth factor–treated flaps had greater volumes than the internal control flap groups and both of the external control flap groups (operation only and blank microspheres). Other intergroup comparisons suggested the absence of a systemic insulin and IGF-1 effect on adiposity. A histomorphometric analysis suggested (1) that insulin and IGF-1 treatment does not alter flap cell composition and (2) that flap augmentation is secondary to the stimulation of cell proliferation and adipocytic differentiation rather than the hypertrophy of mature adipocytes. Further evidence in favor of cell proliferation and differentiation was the discovery of nonanatomic, ectopic fat islands on the pedicle sheath of the treated flaps and the lack of variation in cell size distribution among groups. The authors concluded that the long-term local delivery of insulin and IGF-1 with PLGA/PEG microspheres is an effective method of adipofascial flap augmentation; this method increases the number of mature adipocytes rather than increasing the size of preexisting cells. (Plast. Reconstr. Surg. 106: 373, 2000.)

Safe injection of cultured Schwann cells into peripheral nerve allografts

The effects of cultured host Schwann cells on axonal regeneration in peripheral nerve allografts were studied. Fischer rats served as recipient animals and Buffalo rats provided nerve allografts. Animals were randomized into 9 groups. Rats receiving tibial nerve isografts were left untreated (group I), or injected with isogeneic Fischer Schwann cells (group II) or placebo suspension (group III). Allografts obtained from Buffalo rats were left untreated (group IV), or received isogeneic Fischer Schwann cells (group V), 2 mg/kg Cyclosporin A and Fischer Schwann cells (group VI), 5 mg/kg Cyclosporin A (group VII), or 5 mg/kg Cyclosporin A with Schwann cells (group VIII). No Schwann cell tumors were identified 4 or 8 weeks postoperatively. Group IX animals, harvested 3 days postoperatively, demonstrated no evidence of injection injury. Schwann cells modestly improved axonal regeneration in both isografts and allografts and may have a clinical role in the treatment of peripheral nerve allografts.

Anti-CD40 ligand antibody permits regeneration through peripheral nerve allografts in a nonhuman primate model.

Systemic immunosuppression is typically required to prevent allograft rejection. Antibody-based therapies that induce immune unresponsiveness represent an appealing alternative to nonspecific immunosuppression, which is often associated with significant morbidity. In mice, successful prevention of nerve allograft rejection has been demonstrated through interference with the CD40/CD40 ligand interaction. This study investigated the effectiveness of anti-CD40 ligand monoclonal antibody as single-agent therapy in preventing rejection and supporting nerve regeneration across long nerve allografts in nonhuman primates. Twelve outbred cynomolgus macaques were arranged into six genetically mismatched pairs, with each animal receiving a 5-cm ulnar nerve allograft in the right arm and a 5-cm autograft in the left arm. Mixed lymphocyte reaction assays were used to assess resulting immune unresponsiveness. Treated animals (n = 10) received anti-CD40 ligand monoclonal antibody 10 mg/kg one time, locally applied, and 20 mg/kg systemically on postoperative days 0, 1, 3, 10, 18, and 28, and then monthly. Untreated animals (n = 2) served as the untreated controls. At 4 or 6 months after transplantation, nerves were harvested for histological analysis. Four treated animals underwent an additional challenge after cessation of anti-CD40 ligand monoclonal antibody therapy and nerve graft harvests. Autogenous and allogeneic skin and nerve inlay grafting was performed to assess the permanence of immune unresponsiveness induced by anti-CD40 ligand monoclonal antibody. Animals that received anti-CD40 ligand monoclonal antibody demonstrated robust regeneration across nerve allografts, similar to that seen in the autograft control in the contralateral arm. The histomorphometric analysis of allografts in the untreated animals demonstrated significantly worse measurements compared with their matched autograft controls. Animals that received anti-CD40 ligand monoclonal antibody with concomitant skin allografts had virtually no evidence of nerve regeneration through allografts. Allogeneic skin and nerve allografts applied 2 to 12 months after withdrawal of anti-CD40 ligand monoclonal antibody therapy were consistently rejected. This study demonstrates that anti-CD40 ligand monoclonal antibody prevents rejection and allows regeneration of peripheral nerve allografts in nonhuman primates. The effect of anti-CD40 ligand monoclonal antibody appears to be transient, however, with restoration of immunocompetence shortly after withdrawal of therapy.

Bone deposition/generation with lefort III (midface) distraction

Background: It is essential to critically assess bone deposition in midface distraction. The aim of this study was to characterize the quality and volume of bone deposition at specific osteotomy sites following midface distraction. Methods: At approximately 6 months after distraction, computed tomographic scans with three-dimensional reconstruction were obtained on 10 craniosynostosis syndromal patients who had undergone LeFort III osteotomy and midface distraction. Patient age ranged from 37 to 109 months (mean, 63.7 months) and the distractions ranged from 7 to 15 mm. Both the reconstructed scans and axial cuts were independently evaluated by four blinded observers (two plastic surgeons, an orthodontist, and a radiologist) and graded for bone deposition in predetermined anatomical sites correlated to the osteotomy. Results: The authors found that variable bony bridging occurred at all sites along the osteotomy, but bone deposition was most reliably seen at the pterygomaxillary buttress and nasofrontal junction. In addition, the medial orbital walls tended to show greater consistency in bone deposition than the lateral orbital walls, and deposition at the zygomatic arches was shown to be least likely to occur. The technique of evaluation and the clinically significant findings are discussed. Conclusions: Bony deposition occurs more reliably in the medial facial skeleton following LeFort III osteotomy, and osteotomy through the zygomatic body was more likely to result in deposition than one through the arch.