* Tissue Engineering Strategies to Improve Osteogenesis in the Juvenile Swine Alveolar Cleft Model.

Alveolar (gumline) clefts are the most common congenital bone defect in humans, affecting 1 in 700 live births. Treatment to repair these bony defects relies on autologous, cancellous bone transfer from the iliac crest. This harvest requires a second surgical site with increased surgical time associated with potential complications, while providing only limited cancellous bone. Improvements in treatment protocols that avoid these limitations would be beneficial to patients with clefts and other craniofacial bone defects. There have been steady advances in tissue-engineered (TE) solutions for long-bone defects and adult patients, but advances for the pediatric craniofacial skeleton have been slower to emerge. This study utilizes a previously established juvenile swine model with a surgically created, critical size alveolar defect to test the efficacy of umbilical cord (UC) mesenchymal stem cells (MSCs) treatments on nano-microfiber scaffolds. At 1 month after implanting our TE construct, mineralized tissue in the surgical gap was quantified through computed tomography (CT), and histology, and excised tissue was subjected to mechanical testing. Both undifferentiated and predifferentiated (toward an osteogenic lineage) UC MSCs generated bone within the cleft on a scale comparable to iliac crest cancellous bone, as evidenced by histology and CT scans. All of the pigs treated with scaffold/stem cell combinations had mineralized tissue within the defect, although without filling the entire defect. Several of the experimental animals exhibited poor and/or asymmetric maxillary growth 1 month after the initial surgery, especially if the surgical defect was located on the smaller side of an already asymmetric pig. Our results demonstrate that tissue engineering approaches using UC MSCs are a promising alternative for repair of the alveolar cleft. Data in the pig model demonstrate that implanted scaffolds are at least as good as the current gold standard treatment based on harvesting cancellous bone from the iliac crest, regardless of whether the cells seeded on the scaffold are precommitted to an osteogenic fate.

Application of strain and calibration of Förster Resonance Energy Transfer (FRET) emission for in vitro live cell response to cytoskeletal deformation.

Cellular mechanotransduction is an integral part of many crucial physiological processes, but non-invasive tools for quantifying intracellular strain in vivo are not available for complex tissues such as bone. As a first step to address this gap, we have utilized a novel, non-invasive approach to quantify cellular strain in vitro by employing a transfected alpha-actinin Förster Resonance Energy Transfer (FRET) sensor. Following validation experiments, mouse fibroblasts transfected to express FRET sensors were seeded to a silicone membrane and subjected to up to 10% tensile strain mounted on a multi-photon microscope. During tensile strain, fluorescent emission of acceptor (YFP) and donor (CFP) proteins was quantified. YFP/CFP ratio was normalized to the initial baseline (unstretched) ratio for each cell which demonstrates a negative linear correlation between the relative proximity ratio of emission spectra and cell strain, with a mean decrease of 1.017% normalized ratio for every percent strain experienced by the cell. The exciting implications of our findings are that the discovery of the stable correlation between loss of FRET and experimentally applied strain opens intriguing possibilities for future use of this technology with in vivo research, leading to discoveries improving disease treatments in mechanically sensitive tissues such as bone.

Face Masks for Noninvasive Ventilation: Fit, Excess Skin Hydration, and Pressure Ulcers.

BACKGROUND: Pressure ulcers (stages III and IV) are serious safety events (ie, never events). Healthcare institutions are no longer reimbursed for costs to care for affected patients. Medical devices are the leading cause of pediatric pressure ulcers. Face masks for noninvasive ventilation were associated with a high percentage of pressure ulcers at our institution. METHODS: A prospective cohort study investigated factors contributing to pressure ulcer development in 50 subjects using face masks for noninvasive ventilation. Color imaging, 3-dimensional surface imaging, and skin hydration measurements were used to identify early skin compromise and evaluate 3 interventions to reduce trauma: (1) a silicone foam dressing, (2) a water/polyethylene oxide hydrogel dressing, and (3) a flexible cloth mask. A novel mask fit technique was used to examine the impact of fit on the potential for skin compromise. RESULTS: Fifty subjects age 10.4 ± 9.1 y participated with color images for 22, hydration for 34, and mask fit analysis for 16. Of these, 69% had diagnoses associated with craniofacial anomalies. Stage I pressure ulcers were the most common injury. Skin hydration difference was 317 ± 29 for sites with erythema versus 75 ± 28 for sites without erythema (P < .05) and smallest for the cloth mask (P < .05). Fit distance metrics differed for the nasal, oronasal, and face shield interfaces, with threshold distances being higher for the oronasal mask than the others (P < .05). Areas of high contact were associated with skin erythema and pressure ulcers. CONCLUSIONS: This fit method is currently being utilized to select best-fit masks from available options, to identify the potential areas of increased tissue pressure, and to prevent skin injuries and their complications. Improvement of mask fit is an important priority for improving respiratory outcomes. Strategies to maintain normal skin hydration are important for protecting tissue integrity.

Anatomic study of full facial and scalp allografts without cutaneous facial scars.

Conventional reconstructive procedures for face and scalp reconstruction fall short of aesthetic and functional goals because of the unique quality and quantity of facial and scalp soft tissue. The purpose of this cadaver study was to demonstrate the feasibility of a flap design for full face and scalp composite tissue allotransplantation, without cutaneous facial scars. Six fresh human cadavers were dissected with sagittal scalp and mucosal incisions for full face and scalp harvest without cutaneous facial incisions. Sub-galeal and sub-SMAS dissection allowed for inclusion of the external carotid and internal jugular systems. Time of facial-scalp flap harvesting, length of the arterial and venous pedicles, length of sensory nerves (that were included in the facial flaps) and approximate surface area of the flaps were measured. Three of six flaps were transferred to recipient cadavers and the time of transfer was recorded. As a proof of concept, the external carotid arteries of one of six cadavers was flushed to remove clots and perfused with a radio-opaque latex polymer, Microfil (Flow Tech Inc.), to study flap perfusion by X-ray imaging. In the donor cadaver, the mean harvesting time of the total facial-scalp flap was 105 ± 19 minutes. The mean length of the supraorbital, infraorbital, mental and great auricular nerves were 1.3 ± 0.2, 1.3 ± 0.1, 1.3 ± 0.1, and 4.8 ± 0.6 cm, respectively. The mean length of the external carotid artery and external jugular vein were 8.7 ± 0.3 and 9.2 ± 0.4 cm, respectively. The approximate area of the harvested flap was 1063 ± 60 cm(2). In preparation for full face and scalp allotransplantation in humans, this study has demonstrated the feasibility of a full face and scalp flap without visible facial incisions.

Dissection in the subgaleal and subperiosteal plane: implications on scalp wound healing.

Craniofacial reconstruction often involves the use of dissection in the subperiosteal or subgaleal plane to access the cranial vault and facial skeleton. Clinically, physical changes to the periosteal layer and underlying cortex were observed in the re-operative field. This article compares aspects of wound healing, structural integrity of the progenitor periosteal layer, and underlying bony changes when elevating a subgaleal or subperiosteal flap in a Lewis rat calvarial model. Cranial dissection in the subperiosteal or subgaleal plane was performed on 14 Lewis rats. En bloc resection of the calvarium and overlying soft tissue was harvested at days 0, 1, 3, 7, 14, 28, and 56. Samples underwent SEM imaging and were analysed histologically after trichrome and haematoxylin and eosin staining. One sample of native periosteum underwent cellular expansion to determine periosteal cell regenerative capability. Up to 56 days, subperiosteal dissection results in diffuse hypercellularity within the cambial layer (p < 0.001). There are irregular cortical changes at the periosteal interface and increased disorganised bone remodelling at the temporal ridges. Subgaleal dissection did not reveal any underlying bony changes, and cell counts were not significantly different from controls (p < 0.001). Subperiosteal dissection causes structural and cellular changes to the periosteum and underlying bone composition with a possible influence on its regenerative capability.

Variation in Protein and Calorie Consumption Following Protein Malnutrition in Rattus norvegicus.

Catch-up growth rates, following protein malnutrition, vary with timing and duration of insult, despite unlimited access to calories. Understanding changing patterns of post-insult consumption, relative rehabilitation timing, can provide insight into the mechanisms driving those differences. We hypothesize that higher catch-up growth rates will be correlated with increased protein consumption, while calorie consumption could remain stable. As catch-up growth rates decrease with age/malnutrition duration, we predict a dose effect in protein consumption with rehabilitation timing. We measured total and protein consumption, body mass, and long bone length, following an increase of dietary protein at 40, 60 and 90 days, with two control groups (chronic reduced protein or standard protein) for 150+ days. Immediately following rehabilitation, rats' food consumption decreased significantly, implying that elevated protein intake is sufficient to fuel catch-up growth rates that eventually result in body weights and long bone lengths greater or equal to final measures of chronically fed standard (CT) animals. The duration of protein restriction affected consumption: rats rehabilitated at younger ages had more drastic alterations in consumption of both calories and protein. While rehabilitated animals did compensate with greater protein consumption, variable responses in different ages and sex highlight the plasticity of growth and how nutrition affects body form.

Repairing critical-sized rat calvarial defects with progenitor cell-seeded acellular periosteum: a novel biomimetic scaffold.

BACKGROUND: Many types of scaffolds have been used in bone tissue engineering, with none emerging as favorites. We propose the use of acellular periosteum as a biologic scaffold to allow for progenitor cell adherence, migration, and proliferation in vitro and to test the construct in vivo in a rat calvarial defect model. METHODS: Bovine periosteum was processed to remove all antigenic material (RTI Biologics), and its cambial layer was then seeded with adipose-derived stromal cells (ASCs) or periosteal-derived stromal cells (PSCs) and incubated for 14 days. Adherence required a fibronectin coat and was verified for both cell types via scanning electron microscopy and histology. Two 5-mm diameter calvarial defects were created in each of 19 rats. These were filled with xenograft bone chips and covered with acellular periosteum in combination with cells (ASC or PSC), growth factors (vascular endothelial growth factor, bone morphogenetic protein-2, or both), or alone (controls). Rats were killed 56 days postoperatively. Bone deposition was quantified by microcomputed tomography, and viability was determined histologically. Significance was determined through analysis of variance. RESULTS: Acellular allo-periosteum with a fibronectin coat permitted ASC and PSC adherence, migration, and proliferation in vitro. In the rat calvarial defects, the addition of stem cells (P < .001) and growth factors (P < .001) to the acellular periosteum increased de novo bone growth relative to controls. Although the stem cell source did not influence revitalization (P = .242), the combination of growth factors was more effective (P > .001) than either growth factor alone. The interaction indicated that the 2 cell types did not respond equally to growth factors (P = .039). CONCLUSION: Acellular allo-periosteum is a biomimetic scaffold that permits pleuripotent cell adherence, migration, and proliferation in vitro. The combination of acellular periosteum, xenograft bone, stem cells, and growth factors may prove a viable combination for cranial bone tissue engineering.

Porcine allograft mandible revitalization using autologous adipose-derived stem cells, bone morphogenetic protein-2, and periosteum.

BACKGROUND: Critical defects of the craniomaxillofacial region are often treated with vascularized osteocutaneous free flaps. These lengthy operations may be associated with considerable donor-site morbidity and suboptimal functional and aesthetic results. To overcome these issues, this study investigates an engineered vascularized bone flap using allograft bone, adipose-derived stem cells, and recombinant human bone morphogenic protein (rhBMP)-2 and compares two alternative blood supplies. METHODS: Edentulous porcine hemimandibles were commercially sterilized, packed with rhBMP-2-soaked absorbable collagen sponge and autologous, culture-expanded adipose-derived stem cells, and implanted into two locations within 10 pigs: (1) an intercostal-based periosteal envelope (thoracic) and (2) within the rectus abdominis muscle with insertion of the superficial inferior epigastric vascular pedicle into the medullary cavity (abdominal). The constructs were incubated in vivo for 7 to 8 weeks and harvested to assess de novo bone formation. RESULTS: Radiographic, micro-computed tomographic, and histologic assessments of harvested constructs were performed. Abdominal constructs had a thin rim of new, cancellous bone surrounding a fibrotic core with little allograft remaining. Thoracic allografts were absorbed completely and replaced with new, full-thickness, cancellous bone. Calcitic tissue content was significantly higher in thoracic (474.16 +/- 75.93 ml) compared with abdominal (143.20 +/- 46.39 ml) constructs (p < 0.006). New bone in both groups contained Haversian systems, but only thoracic constructs contained marrow elements and blood vessels resembling normal bone. CONCLUSIONS: These data demonstrate revitalization of large-volume allograft bone, and have positive implications for bone tissue engineering. Allograft revitalization in thoracic but not abdominal constructs reinforces the critical role of the periosteum in the process.

Endoscopically assisted Le Fort III osteotomy using an ultrasonic scalpel: a feasibility study in cadavers.

Drawbacks to conventional Le Fort III osteotomy include bleeding, infection, relapse, and scar at the coronal incision. We performed an endoscopically assisted Le Fort III osteotomy with an ultrasonic scalpel in cadavers to develop a new technique that minimizes such complications. Endoscopically assisted Le Fort III osteotomy was performed in 3 fresh, adult human cadavers. Access incisions included the transconjunctival lower lid with lateral canthotomy, the lateral upper gingivobuccal sulcus, and a stab incision in the medial aspect of the upper eyelid. Osteotomies at the zygomaticofrontal suture, the lateral orbital wall, the orbital floor, and the medial orbital wall were carried out with an ultrasonic scalpel under direct and endoscopic visualization from the trasconjunctival incision. The zygomatic arch and the pterygomaxillary region were osteotomized via the upper gingivobuccal sulcus incisions. The nasofrontal junction and the septum were accessed and cut via a stab incision in the medial upper eyelids. Disimpaction was completed with minor digital pressure inferiorly.Each of the 3 Le Fort III osteotomies was complete, and mobility was checked by manual examination. There was a steep learning curve to the operation, but the final cadaver dissection took 99 minutes to complete. The ultrasonic scalpel provided for maximal ease in cutting bone and minimal disruption to adjacent soft tissues as judged by postoperative direct examination. This cadaver study demonstrates the feasibility of a minimally invasive, endoscopically assisted Le Fort III osteotomy using an ultrasonic scalpel. Further experimental work combined with refinements in technique and equipment will help bring this advancement into clinical application.

Endoscopically assisted Le Fort I osteotomy using an ultrasonic scalpel: a feasibility study in cadavers.

PURPOSE: Drawbacks to conventional Le Fort I osteotomy include bleeding, infection, and relapse. The open approach predisposes itself to each of these complications through various means. We performed an endoscopically assisted Le Fort I osteotomy with an ultrasonic scalpel in cadavers to develop a new technique that minimizes such complications. MATERIALS AND METHODS: Endoscopically assisted Le Fort I osteotomy was performed in 4 fresh adult human cadavers. Two 1-cm gingivobuccal sulcus incisions were used to approach the maxilla. Osteotomies were carried out with an ultrasonic scalpel from within the maxillary sinus under endoscopic visualization after a small antrostomy was made in its anterior wall. The external periosteal sleeve to the maxilla remained intact, eliminating the risk of massive hemorrhage and preserving bony vascularity. A stab incision was made with a 2-mm osteotome above the anterior nasal spine to separate the caudal septum from the Le Fort I segment. Pterygomaxillary disjunction was also performed with classic osteotomes. Disimpaction was completed with minor digital pressure inferiorly. RESULTS: Each of the 4 Le Fort I osteotomies was complete, and mobility was checked by manual examination. There was a steep learning curve to the operation, but the final cadaveric dissection took 37 minutes to complete. At no time did the ultrasonic scalpel violate the maxillary periosteum as judged by postoperative direct examination. DISCUSSION: This cadaveric study shows the feasibility of an endoscopically assisted Le Fort I osteotomy by use of an ultrasonic scalpel. Further experimental work combined with refinements in technique and equipment will help bring this advancement into clinical application.