Anthony Spangenberger

The fetal cleft palate: I. Characterization of a congenital model.

Any animal model of a human congenital anomaly established by iatrogenic methods involving intrauterine fetal manipulation has limited clinical applicability. A congenital model that more closely simulates the etiopathogenesis of a human anomaly may provide data that can more readily be extrapolated to that anomaly and, therefore, be used in diagnostic and management strategies. The present work provides a description and characterization of a congenital model of cleft palate in the goat. Palatal shelf closure normally occurs at approximately day 38 of gestation in the caprine species. Sixteen pregnant goats were gavaged twice daily during gestational days 32 through 41 [term, 145 days] with a plant slurry of Nicotiana glauca containing the piperidine alkaloid teratogen anabasine. Gross analysis and measurement of fetal clefts were performed at 60, 70, and 85 days gestation (four fetuses were studied at each time point). Seventeen clefted kids were sacrificed at specific intervals after birth (2 weeks, and 1, 3, and 6 months); after skull debridement and preparation, they were compared with 12 unclefted control kids. Complete clefting of the secondary palate occurred in 97 percent of the fetuses. In all cases, the cleft extended from the posterior aspect of the alveolar ridge to the uvula; the majority of these clefts were bilateral, with complete detachment of the vomer. Morphologically, these clefts were similar to human clefts. Eighteen percent of clefted newborn kids demonstrated gross maxillary hypoplasia and midfacial retrusion at birth with a relative Class III malocclusion. Direct measurement of the congenital caprine skulls confirmed these findings. The incidence of midfacial growth abnormalities in these clefted animals raises questions regarding the etiopathogenesis of facial dysmorphology that is unrelated to scarring of the maxilla. This congenital cleft palate model is currently being used to explore these questions and others related to craniofacial growth and palatal function after in utero repair.

Long-term fate of transplanted autologous fat in a novel rabbit facial model.

Background: The concept of fat transplantation is not a new one in plastic surgery. Studies assessing fat graft viability following transplantation, however, have yielded conflicting observations. The present study evaluated the long-term fate of transplanted autologous fat using a novel facial augmentation model the authors have developed in the rabbit. Methods: Thirteen adult New Zealand White rabbits underwent fat harvest using a modified Coleman technique. The left upper lip was used as the recipient site; the right upper lip remained unaltered in each case. After harvest from the groin, 1 ml of adipose tissue was transplanted to the left upper lip of each rabbit. Rabbits were euthanized 1, 3, 6, and 12 months after unilateral lip augmentation. Coronal sections of both upper lips were evaluated histologically; the right upper lip served as an internal control in each case. Results: Histologic evaluation of specimens harvested at all postoperative time points demonstrated viable, transplanted fat graft in each case. Use of the nonoperated right lip allowed for paired evaluation of the experimental and control sides. A significant inflammatory response was present in the early phases, with evidence of neovascularization. Transplanted fat tissue was clearly visible in rabbits harvested 1 year postoperatively. Conclusions: The authors have established a novel, reproducible model with an internal control for long-term evaluation of transplanted fat. Histologic examination suggests an early inflammatory response to the injected fat followed by sequestration of nonviable tissue. The transplanted fat remained viable at 1 year, with good overall survivability and minimal fibrosis.

The fetal cleft palate: II. Scarless healing after in utero repair of a congenital model.

The role of fetal surgery in the treatment of non-life-threatening congenital anomalies remains a source of much debate. Before such undertakings can be justified, models must be established that closely resemble the respective human anomalies, and the feasibility and safety of these in utero procedures must be demonstrated. The authors recently described and characterized a congenital model of cleft palate in the goat. The present work demonstrates the methodology they developed to successfully repair these congenital cleft palates in utero, and it shows palatal healing and development after repair. A surgically created cleft model was developed for comparative purposes. Palatal shelf closure normally occurs at approximately day 38 of gestation in the caprine species. Six pregnant goats were gavaged twice daily during gestational days 32 to 41 (term, 145 days) with a plant slurry of Nicotiana glauca containing the piperidine alkaloid anabasine; the 12 fetuses had complete congenital clefts of the secondary palate. Repair of the congenital clefts was performed at 85 days of gestation using a modified von Langenbeck technique employing lateral relaxing incisions with elevation and midline approximation of full-thickness, bilateral, mucoperiosteal palatal flaps followed by single-layer closure. Six congenitally clefted fetuses underwent in utero repair, six remained as unrepaired controls. Twelve normal fetuses underwent surgical cleft creation by excision of a 20 x 3 mm full-thickness midline section of the secondary palate extending from the alveolus to the uvula, at 85 days of gestation. Six surgically clefted fetuses underwent concurrent repair of the cleft at that time; six clefted fetuses remained as unrepaired controls. At 2 weeks of age, no congenitally or surgically created clefts repaired in utero demonstrated gross or histologic evidence of scar formation. A slight indentation at the site of repair was the only remaining evidence of a cleft. At 6 months of age, normal palatal architecture, including that of mucosal, muscular, and glandular elements, was seen grossly and histologically. Cross-section through the mid-portion of the repaired congenitally clefted palates demonstrated reconstitution of a bilaminar palate, with distinct oral and nasal mucosal layers, after single-layer repair. In utero cleft palate repair is technically feasible and results in scarless healing of the mucoperiosteum and velum. The present work represents the first in utero repair of a congenital cleft palate model in any species. The use of a congenital cleft palate model that can be consistently reproduced with high predictability and little variation represents the ideal experimental situation. It provides an opportunity to manipulate specific variables, assess the influence of each change on the outcome and, subsequently, extrapolate such findings to the clinical arena with a greater degree of relevance.

The Fetal Cleft Palate : V. Elucidation of the Mechanism of Palatal Clefting in the Congenital Caprine Model

Background: Failure of palatal shelf fusion at the precise gestational time point when shelf elevation and migration normally occur results in clefting. The present study defined the mechanism of clefting in the congenital caprine model by evaluating the temporal sequence of palatal shelf fusion. Methods: Six Spanish-type goats pregnant with twins were gavaged for 10 days (gestational days 32 to 41) with anabasine. Goats were examined with ultrasound throughout this period to assess fetal movement and evaluated histologically. Twelve untreated, unclefted fetuses served as controls. Results: After anabasine induction, real-time ultrasound confirmed a dramatic decrease in fetal movement. Hyperflexion of the neck occurred throughout the experimental period compared with control fetuses, in which spontaneous neck movements occurred. Histologic evaluation of the induced fetuses demonstrated the tongue wedged between the palatal shelves throughout the period of fusion. The shelves remained vertically oriented without elevation, ultimately resulting in clefting. The unclefted, control group demonstrated progressive elevation and migration of the palatal shelves between gestational days 38 and 40, with contact and fusion of the medial edge epithelia in the midline. Conclusions: Palatal clefting in the congenital caprine model occurred concomitant with reduced fetal movement. Resultant neck hyperflexion causes the tongue to obstruct medial migration of the palatal shelves and fusion, whereas the shelves themselves are either directly prevented from elevation secondary to anabasine impairment or indirectly secondary to the obstructing tongue. Although the molecular mechanism for the teratogenic effects of anabasine remains elusive, the authors have demonstrated an association between reduced fetal movement and palatal clefting in their congenital caprine model.

Effect of Sustained-Release PDGF and TGF-β on Cyclophosphamide-Induced Impaired Wound Healing

Background: Proper wound healing is pivotal to successful surgical outcomes. Previous studies have shown that growth factors can be used to enhance tissue repair under impaired healing conditions. However, because of limited delivery methods, the growth factors in these studies were delivered either topically or as a single local administration. Methods: Sixty Sprague-Dawley rats were divided equally into five groups and served as untreated normal controls or were implanted subcutaneously with a novel sustained-release drug delivery system through a dorsal incisional wound. This system delivered either transforming growth factor (TGF)-ß alone, platelet-derived growth factor (PDGF) alone, or TGF-ß and PDGF in combination, or served as unloaded sham controls. Wound healing was impaired in all treated rats by the administration of cyclophosphamide on days 1, 3, and 5. Wound tensile breaking strength was determined on days 4, 7, and 14. Results: Sustained release of either TGF-ß or PDGF alone not only failed to improve the healing of cyclophosphamide-induced impaired wound healing but resulted in a paradoxical decrease in wound tensile breaking strength by day 7. However, the combined delivery of both TGF-ß and PDGF improved wound healing and significantly increased wound tensile breaking strength by day 7. Conclusions: Sustained-release delivery of TGF-ß and PDGF in combination, but not separately, by a subcutaneously implanted drug delivery system significantly improves cyclophosphamide-induced impaired wound healing in rats.

Role of TGF-β and FGF in the Treatment of Radiation-Impaired Wounds Using a Novel Drug Delivery System

Background: Despite refinements in radiotherapy, radiation-impaired wound healing continues to be a major source of postoperative morbidity with few treatment options. The application of polypeptide growth factors has been investigated in both the clinical and laboratory settings. The authors used a novel sustained-release delivery system to examine the effect of transforming growth factor (TGF)-&bgr; and fibroblast growth factor (FGF) on radiation-impaired wound healing in a rodent model. Methods: Eighty Sprague-Dawley rats underwent dorsal skin surface irradiation of 2500 cGy using a medical linear accelerator producing energy of 6 MeV followed by creation of a full-thickness skin incision. Six groups of 16 animals underwent either sham irradiation (irradiation control); irradiation only; irradiation and unimpregnated delivery system only; or irradiation and either TGF-&bgr;, FGF, or TGF-&bgr; plus FGF combined. Four animals from each group were euthanized at 4, 7, 14, and 28 days, and the harvested specimens underwent ultimate tensile strength testing and histologic evaluation. Results: All five irradiated groups had significantly lower ultimate tensile strength than the sham-irradiated control group at all time points (p < 0.05), thus validating the authors’ model of radiation-impaired wound healing. Functional analysis demonstrated that all three growth factor–treated groups had significantly higher tensile strengths than either of the untreated irradiated groups at 14 days after wounding (p < 0.05). Histologic evaluation of the irradiated groups revealed increased cellularity and more organized collagen architecture of all treated groups when compared with the untreated groups, with the most pronounced differences seen at 7 days and 14 days after wounding. Conclusions: This study effectively demonstrates that TGF-&bgr; and FGF act individually and synergistically when delivered locally by means of a sustained release system to improve ultimate tensile strength in an acute postirradiation impaired wound-healing model.

Investigation of the growth and metastasis of malignant melanoma in a murine model: the role of supplemental vitamin A.

Vitamin A possesses both wound-healing and antitumor actions. Vitamin A–induced fibroplasia results in subsequent increased collagen production and deposition. This effect of vitamin A has been shown to result in the production of collagenous capsules around several murine breast and lung tumor systems. This tumor encapsulation process can potentially convert a systemic disease to a local one that can be easily treated by tumor excision. The goal of the present study was to determine whether supplemental vitamin A could promote the encapsulation of a murine melanoma. Sixty DBA/2J male mice were inoculated intracutaneously with 1 × 106 Cloudman S91 melanoma cells using a 30-gauge needle. The mice were divided into three groups: a control group, a pre-vitamin A group, and a post-vitamin A group. The control mice were fed a commercial chow containing 15,000 IU of vitamin A and 6.4 mg of beta-carotene per kilogram diet, considerably more than the National Research Council’s recommended daily allowance of vitamin A for normal mice. The control diet was, therefore, not vitamin A–deficient. The pre-vitamin A mice were fed the basal chow supplemented with 150,000 IU of vitamin A per kilogram diet for 10 days before inoculation and for the remainder of the study. The post-vitamin A mice were fed the vitamin A–supplemented diet beginning on the day of inoculation and continuing for the remainder of the study. Sixty days after inoculation, tumor growth was assessed and the five mice remaining in each group were euthanized. Ventral skin at the site of inoculation was harvested for histologic assessment of local tumor growth and invasiveness. The liver and lungs of each of these mice were also harvested for histologic assessment of tumor metastasis. Sixty days after tumor inoculation, a 60 percent survival rate was observed in the control group as opposed to the vitamin A–supplemented animals, which demonstrated a 100 percent survival rate in both groups (n = 5 in each group). Decreased mean tumor size and gross tumor in most vitamin A–supplemented animals were statistically significant when compared with the control animals. The control animals had a mean tumor size of 26.1 mm, whereas the post-vitamin A group had a mean tumor size of 5.7 mm. One hundred percent of the control group exhibited tumor; one animal had distant metastases. The pre-vitamin A group did not exhibit any tumor growth, and the post-vitamin A group exhibited tumor growth in 40 percent of animals. Neither vitamin A–supplemented group showed any evidence of distant metastases. The animals supplemented with vitamin A demonstrated decreased tumor growth and metastasis. This in vivo model may indicate a potential prophylactic and therapeutic role for supplemental vitamin A in the treatment of malignant melanoma.

Osteochondral reconstruction of a non-weight-bearing joint using a high-density porous polyethylene implant.

Currently, there is no reliable reconstructive modality allowing anatomic resurfacing of traumatic digital osteochondral articular defects. The purpose of the present study is to demonstrate the utility of Medpor, a high-density porous polyethylene (HDPP) scaffold biomaterial that can (1) be readily contoured to fit any joint defect, (2) permit stable internal fixation, and (3) permit osteocyte and chondrocyte ingrowth and subsequent articular cartilage resurfacing necessary to restore joint congruity. HDPP has gained wide acceptance for use in craniofacial and skeletal reconstruction and augmentation. An avian non-weight-bearing joint model was designed to study the role of the HDPP implant in small joint reconstruction. An osteochondral defect was created with a 5-mm circular punch in the humeral articular surface of both glenohumeral joints of 32 adult White Leghorn chickens. In each animal, one defect was press-fitted with a correspondingly sized HDPP implant (HDPP implant group); the contralateral defect was filled with the original osteochondral plug (isograft group) or left unrepaired (control group). At 2 weeks, and 1, 3, and 6 months,joints from each group were harvested and evaluated. Over the 6-month study period, joints in the control group demonstrated healing with dense collagenous scar tissue leaving residual defects at the articular surfaces and significant degenerative disease of the glenohumeral joints radiographically. Joints in the isograft group demonstrated near-complete resorption with some preservation of the cartilaginous cap but overall depression of the articular surface and significant degenerative joint disease. Joints in the HDPP implant group demonstrated stable fixation by highly mineralized bony trabecular ingrowth, preservation of the articular contour of the humeral head, and no evidence of significant degenerative joint disease. These findings indicate a potential role for this high-density porous polyethylene implant in the reconstruction of small joint articular and osseous defects.