Stephen E. Feinberg

Mechanical and in vivo performance of hydroxyapatite implants with controlled architectures

Internal architecture has a direct impact on the mechanical and biological behaviors of porous hydroxyapatite (HA) implant. However, traditional processing methods provide minimal control in this regard. To address the issue, we developed a new processing method combining image-based design and solid free-form fabrication. We have previously published the processing method showing fabricated HA implants and their chemical properties. This study characterized the mechanical and the in vivo performance of designed HA implants. Thirteen HA implants with orthogonal channels at 40% porosity were tested on an Instron machine. The compressive strength and compressive modulus measured were 30+/-8 MPa and 1.4+/-0.4 GPa, comparable to coralline porous HA. Twenty-four cylindrical HA implants with two architecture designs, orthogonal and radial channels, were implanted in the mandibles of four Yucatan minipigs for 5 and 9 weeks. Normal bone regeneration occurred in both groups. At 9 weeks, bone penetrated 1.4mm into both scaffold designs. The percent bone ingrowth in the penetration zone was higher in the orthogonal channel design but not statistically different due to the low number of samples. However, the overall shape of the regenerated bone tissue was significantly different. In the orthogonal design, bone and HA formed an interpenetrating matrix, while in the radial design, the regenerated bone formed an intact piece at the center of the implant. These preliminary results showed that controlling the overall geometry of the regenerated bone tissue is possible through the internal architectural design of the scaffolds.

Hydroxyapatite implants with designed internal architecture

Porous hydroxyapatite (HA) has been used as a bone graft material in the clinics for decades. Traditionally, the pores in these HAs are either obtained from the coralline exoskeletal patterns or from the embedded organic particles in the starting HA powder. Both processes offer very limited control on the pore structure. A new method for manufacturing porous HA with designed pore channels has been developed. This method is essentially a lost-mold technique with negative molds made with Stereolithography and a highly loaded curable HA suspension as the ceramic carrier. Implants with designed channels and connection patterns were first generated from a Computer-Aided-Design (CAD) software and Computer Tomography (CT) data. The negative images of the designs were used to build the molds on a stereolithography apparatus with epoxy resins. A 40 vol% HA suspension in propoxylated neopentyl glycol diacrylate (PNPGDA) and iso-bornyl acrylate (IBA) was formulated. HA suspension was cast into the epoxy molds and cured into solid at 85 °C. The molds and acrylate binders were removed by pyrolysis, followed by HA green body sintering. With this method, implants with six different channel designs were built successfully and the designed channels were reproduced in the sintered HA implants. The channels created in the sintered HA implants were between 366 μm and 968 μm in diameter with standard deviations of 50 μm or less. The porosity created by the channels were between 26% and 52%. The results show that HA implants with designed connection pattern and well controled channel size can be built with the technique developed in this study.

Development and Characterization of a Tissue-engineered Human Oral Mucosa Equivalent Produced in a Serum-free Culture System

A problem maxillofacial surgeons face is a lack of sufficient autogenous oral mucosa for reconstruction of the oral cavity. Split-thickness or oral mucosa grafts require more than one surgical procedure and can result in donor site morbidity. Skin has disadvantages of adnexal structures and a different keratinization pattern than oral mucosa. In this study, we successfully assembled, ex vivo, a human oral mucosa equivalent, consisting of epidermal and dermal components, in a defined, essential-fatty-acid-deficient, serum-free culture medium without a feeder layer, that could be used for intra-oral grafting in humans. Autogenous oral keratinocytes were seeded onto a cadaveric dermis, AlloDerm™. The oral mucosa equivalent was cultured at an air-liquid interface for 2 wks. The resulting equivalent had a well-stratified parakeratinized epithelial layer similar to native oral keratinized mucosa. Expression of differentiation markers, filaggrin and cytokeratin 10/13, suggested a premature keratinized state. The presence of proliferation markers, proliferating cell nuclear antigen (PCNA) and Ki-67, suggested a state of hyperproliferation. Fatty acid composition of the equivalent was similar to that of in vitro cultured oral keratinocytes but differed from the that of in vivo native tissue, showing a lower content of 18:2 and 20:4, and a higher content of 16:1 and 18:1 fatty acids, respectively. The keratinocytes of the equivalent appeared to be in a more active and proliferative state than native keratinized mucosa. The dynamic nature of the cell population on the oral mucosa equivalent may be beneficial for intra-oral grafting procedures and for transfection of the keratinocytes.

Ex vivo development of a composite human oral mucosal equivalent

PURPOSE The aim of this study was the ex vivo development of a composite oral mucosal equivalent composed of a continuous stratified layer of human oral keratinocytes grown on a cadaveric human dermal matrix in a defined medium without a feeder layer. MATERIALS AND METHODS Enzymatically dissociated human oral keratinocytes from keratinized oral mucosa were cultured, submerged in a serum-free, low-calcium (0.15 mmol/L) supplemented medium, and expanded through several passages. Once a sufficient population of keratinocytes was reached, they were seeded on 1-cm2 pieces of AlloDerm (LifeCell Co, Woodlands, TX), an acellular nonimmunogenic cadaveric human dermis, at cell densities of 2.5 X 10(4), 5.0 X 10(4), 1.25 X 10(5), or 2.5 X 10(5). The oral keratinocyte-AlloDerm composites were cultured while submerged in a high-calcium (1.8 mmol/L) medium for 4 days. After 4 days, the composites were raised to an air-liquid interface. Samples of the composites were taken for histologic examination at 4, 11, and 18 days postseeding of the keratinocytes on the AlloDerm. RESULTS At day 4, only the seeded cell density of 2.5 X 10(5) cells/cm2 formed a continuous monolayer on the AlloDerm. At day 11, a continuous stratified epithelium was seen, and at day 18 a well-differentiated, confluent parakeratotic epithelial layer was developed at cell densities of 5.0 X 10(4), 1.25 X 10(5), and 2.5 X 10(5)cells/cm2. CONCLUSION With the method used, it was possible to successfully develop an ex vivo composite oral mucosal equivalent that consisted of a stratified epidermis on a dermal matrix.

Development of a Tissue-Engineered Human Oral Mucosa: From the Bench to the Bed Side

The main objective of this publication is to make the reader aware of the complexity and steps that are necessary to make a Food and Drug Administration (FDA)-approved laboratory produced cell-based device, for use in clinical trials for reconstructive surgery. Most tissue-engineered cell-based devices are considered as ‘human somatic cell therapy’ and fall under the auspices of the Center of Biologic Evaluation and Research (CBER) and are considered a combination product by the FDA. We have illustrated the algorithm that is necessary to follow an Independent New Drug (IND) application by using our ex vivo produced oral mucosa equivalents (EVPOME), a tissue-engineered oral mucosa, as an example of a cell-based device that needs FDA approval prior to clinical application. By illustrating the experimental approach and presenting resulting data we attempt to explain each step that we address along the way.

Isolation of Human Oral Keratinocyte Progenitor/Stem Cells

Progenitor/stem cell populations of epithelium are known to reside in the small-sized cell population. Our objective was to physically isolate and characterize an oral keratinocyte-enriched population of small-sized progenitor/stem cells. Primary human oral mucosal keratinocytes cultured in a chemically defined serum-free culture system, devoid of animal-derived feeder cells, were sorted by relative cell size and characterized by immunolabeling for β1 integrin, nuclear transcription factor, peroxisome proliferator-activated receptor-gamma, and cell-cycle analysis. Sorted cells were distinguished as progenitor/stem cells by functional assays and their ability to regenerate an oral mucosal graft. Small-sized cells demonstrated the lowest expression of peroxisome proliferator-activated receptor-gamma, the highest colony-forming efficiency, a longer long-term proliferative potential, an enriched quiescent cell population, and the ability to regenerate an oral mucosal graft, implying that the small-sized cultured oral keratinocytes contained an enriched population of progenitor/stem cells.

Manufacturing and Characterization of 3‐D Hydroxyapatite Bone Tissue Engineering Scaffolds

Abstract: Internal architecture has a direct impact on the mechanical and biological behaviors of porous hydroxyapatite (HA) implants. However, traditional processing methods provide very minimal control in this regard. This paper reviews a novel processing technique developed in our laboratory for fabricating scaffolds with controlled internal architectures. The preliminary mechanical property and in vivo evaluation of these scaffolds are also presented.

Image-Based Biomimetic Approach to Reconstruction of the Temporomandibular Joint

This article will present an image-based approach to the designing and manufacturing of biomimetic tissue engineered temporomandibular (TMJ) condylar prosthesis. Our vision of a tissue-engineered TMJ prosthesis utilizes a 3-D designed and manufactured biodegradable scaffold shaped similar to a condylar head and neck, i.e. a condylar-ramus unit (CRU). The fabricated CRU scaffold can be constructed with a specific intra-architectural design such that it will enhance the formation of tissue from implanted cells placed within its interstices. These biologic cues could influence scaffold-implanted mesenchymal stem cells (MSC) or bone marrow stromal cells (BMSC) to form a fibrocartilaginous joint surface, or cap, on top of a bony strut, similar to a costochondral rib graft (CCRG), which could be fixed to the mandibular ramus. This new approach to tissue engineering a TMJ would be advantageous because of its patient site-specific anatomical configuration as well as its potential ability to adapt to the loading forces placed on it during function.

Evaluation of Transplanted Tissue-Engineered Oral Mucosa Equivalents in Severe Combined Immunodeficient Mice

The aim of this study was to determine the optimal stage of development at which transplant human ex vivo-produced oral mucosa equivalents (EVPOMEs) in vivo. EVPOMEs were generated in a serum-free culture system, without the use of an irradiated xenogeneic feeder layer, by seeding human oral keratinocytes onto a human cadaveric dermal equivalent, AlloDerm. EVPOMEs were cultured for 4 days submerged and then for 7 or 14 days at an air-liquid interface to initiate stratification before transplantation into SCID mice. AlloDerm, without epithelium, was used as a control. Mice were killed on days 3, 10, and 21 posttransplantation. Epithelium of the transplanted EVPOMEs was evaluated with the differentiation marker keratin 10/13. Dermal microvessel ingrowth was determined by immunohistochemistry with a mouse vascular marker, lectin binding from Triticum vulgaris. The presence and stratification of the epithelium were correlated with revascularization of the underlying dermis. The microvessel density of AlloDerm without epithelium was less than that of EVPOMEs with an epithelial layer. Microvessel density of the dermis varied directly with the degree of epithelial stratification of the EVPOMEs. The EVPOMEs cultured at an air-liquid interface for 7 days had the optimal balance of neoangiogenesis and epithelial differentiation necessary for in vivo grafting.

In vitro comparison of parameters affecting the fixation strength of sagittal split osteotomies

PURPOSE The goal of this study was to determine how different parameters affect the bending strength of human cadaver mandibles that have undergone a sagittal split osteotomy. MATERIALS AND METHODS The effects of screw material (titanium [Ti] vs polylactic acid/polyglycolic acid [PLA/PGA]), screw configuration (linear vs inverted L-shape), screw diameter (2.0 mm vs 2.7 mm), material into which screws were inserted (human mandible, bovine rib, synthetic polymer), and loading rate (1.0 mm/min vs 10.0 mm/min) were quantified. Also, biomechanical principles were used to model shear stress and displacement. Variable lever arms, screw material, screw diameter, screw configuration, distance between screws, and bone properties were all evaluated in this model. RESULTS Accounting for variable mandible geometries and differentiating between deflections (and shear stresses) due to bending and due to torsion, in vitro mechanical testing revealed that there was a statistically significant difference in total shear stress at 3 mm of deflection depending on screw material (Ti > PLA/PGA), screw diameter, and material into which screws are inserted (mandibles > ribs = synthetic polymer). There was no significant difference in total shear stress depending on screw configuration or strain rate. CONCLUSION Total shear stress and deflections are important and more viable parameters than load to assess parameters of clinical importance in osteotomy or fracture fixation.