Bone Flap Resorption in Pediatric Patients Following Autologous Cranioplasty.

Following a decompressive craniectomy, the autologous bone flap is generally considered the reconstructive material of choice in pediatric patients. Replacement of the original bone flap takes advantage of its natural biocompatibility and the associated low risk of rejection, as well as the potential to reintegrate with the adjacent bone and subsequently grow with the patient. However, despite these advantages and unlike adult patients, the replaced calvarial bone is more likely to undergo delayed bone resorption in pediatric patients, ultimately requiring revision surgery. In this review, we describe the materials that are currently available for pediatric cranioplasty, the advantages and disadvantages of autologous calvarial replacement, the incidence and classification of bone resorption, and the clinical risk factors for bone flap resorption that have been identified to date.

Simple signaling molecules for inductive bone regenerative engineering.

With greater than 500,000 orthopaedic procedures performed in the United States each year requiring a bone graft, the development of novel graft materials is necessary. We report that some porous polymer/ceramic composite scaffolds possess intrinsic osteoinductivity as shown through their capacity to induce in vivo host osteoid mineralization and in vitro stem cell osteogenesis making them attractive synthetic bone graft substitutes. It was discovered that certain low crystallinity ceramics partially dissociate into simple signaling molecules (i.e., calcium and phosphate ions) that induce stem cells to endogenously produce their own osteoinductive proteins. Review of the literature has uncovered a variety of simple signaling molecules (i.e., gases, ions, and redox reagents) capable of inducing other desirable stem cell differentiation through endogenous growth factor production. Inductive simple signaling molecules, which we have termed inducerons, represent a paradigm shift in the field of regenerative engineering where they can be utilized in place of recombinant protein growth factors.

VEGF-incorporated biomimetic poly(lactide-co-glycolide) sintered microsphere scaffolds for bone tissue engineering.

Regenerative engineering approaches utilizing biomimetic synthetic scaffolds provide alternative strategies to repair and restore damaged bone. The efficacy of the scaffolds for functional bone regeneration critically depends on their ability to induce and support vascular infiltration. In the present study, three-dimensional (3D) biomimetic poly(lactide-co-glycolide) (PLAGA) sintered microsphere scaffolds were developed by sintering together PLAGA microspheres followed by nucleation of minerals in a simulated body fluid. Further, the angiogenic potential of vascular endothelial growth factor (VEGF)-incorporated mineralized PLAGA scaffolds were examined by monitoring the growth and phenotypic expression of endothelial cells on scaffolds. Scanning electron microscopy micrographs confirmed the growth of bone-like mineral layers on the surface of microspheres. The mineralized PLAGA scaffolds possessed interconnectivity and a compressive modulus of 402 ± 61 MPa and compressive strength of 14.6 ± 2.9 MPa. Mineralized scaffolds supported the attachment and growth and normal phenotypic expression of endothelial cells. Further, precipitation of apatite layer on PLAGA scaffolds resulted in an enhanced VEGF adsorption and prolonged release compared to nonmineralized PLAGA and, thus, a significant increase in endothelial cell proliferation. Together, these results demonstrated the potential of VEGF-incorporated biomimetic PLAGA sintered microsphere scaffolds for bone tissue engineering as they possess the combined effects of osteointegrativity and angiogenesis.

Tissue-engineered matrices as functional delivery systems: adsorption and release of bioactive proteins from degradable composite scaffolds.

A tissue-engineered bone graft should imitate the ideal autograft in both form and function. However, biomaterials that have appropriate chemical and mechanical properties for grafting applications often lack biological components that may enhance regeneration. The concept of adding proteins such as growth factors to scaffolds has therefore emerged as a possible solution to improve overall graft design. In this study, we investigated this concept by loading porous hydroxyapatite-poly(lactide-co-glycolide) (HA-PLAGA) scaffolds with a model protein, cytochrome c, and then studying its release in a phosphate-buffered saline solution. The HA-PLAGA scaffold has previously been shown to be bioactive, osteoconductive, and to have appropriate physical properties for tissue engineering applications. The loading experiments demonstrated that the HA-PLAGA scaffold could also function effectively as a substrate for protein adsorption and release. Scaffold protein adsorptive loading (as opposed to physical entrapment within the matrix) was directly related to levels of scaffold HA-content. The HA phase of the scaffold facilitated protein retention in the matrix following incubation in aqueous buffer for periods up to 8 weeks. Greater levels of protein retention time may improve the protein's effective activity by increasing the probability for protein-cell interactions. The ability to control protein loading and delivery simply via composition of the HA-PLAGA scaffold offers the potential of forming robust functionalized bone grafts.

Biodegradable polyphosphazene-nanohydroxyapatite composite nanofibers: scaffolds for bone tissue engineering.

Bone is a natural composite comprised of hierarchically arranged collagen fibrils, hydroxyapatite and proteoglycans in the nanometer scale. This preliminary study reports the fabrication of biodegradable poly[bis(ethyl alanato)phosphazene]-nanohydroxyapatite (PNEA-nHAp) composite nanofiber matrices via electrospinning. Binary solvent compositions of THF and ethanol were used as a spinning solvent to attain better nanohydroxyapatite dispersibility in PNEA solution. These nanocomposites were characterized for morphology, nHAp distribution and content using spectroscopy and gravimetric estimations. Composite nanofibers fabricated in the diameter range of 100-310 nm could encapsulate 20-40 nm nHAp crystals. A better composite nanofiber yield was obtained for 50% (w/w) nHAp experimental loadings. Incremental experimental loading beyond 60% (w/w) hindered electrospinning due to polymer-nHAp phase separation. Composites nanofibers had a rougher surface and nodules along the length of the fibers suggesting nHAp encapsulation. Further, characterization via energy dispersive X-ray spectroscopy and X-ray mapping confirmed the nHAp encapsulation. Providing cells with a natural bone like environment with a fibrillar structure and natural hydroxyapatite can enhance bone tissue regeneration/repair.

Induction of angiogenesis in tissue-engineered scaffolds designed for bone repair: a combined gene therapy-cell transplantation approach.

One of the fundamental principles underlying tissue engineering approaches is that newly formed tissue must maintain sufficient vascularization to support its growth. Efforts to induce vascular growth into tissue-engineered scaffolds have recently been dedicated to developing novel strategies to deliver specific biological factors that direct the recruitment of endothelial cell (EC) progenitors and their differentiation. The challenge, however, lies in orchestration of the cells, appropriate biological factors, and optimal factor doses. This study reports an approach as a step forward to resolving this dilemma by combining an ex vivo gene transfer strategy and EC transplantation. The utility of this approach was evaluated by using 3D poly(lactide-co-glycolide) (PLAGA) sintered microsphere scaffolds for bone tissue engineering applications. Our goal was achieved by isolation and transfection of adipose-derived stromal cells (ADSCs) with adenovirus encoding the cDNA of VEGF. We demonstrated that the combination of VEGF releasing ADSCs and ECs results in marked vascular growth within PLAGA scaffolds. We thereby delineate the potential of ADSCs to promote vascular growth into biomaterials.

Amorphous hydroxyapatite-sintered polymeric scaffolds for bone tissue regeneration: physical characterization studies.

Given the inherent shortcomings of autografts and allografts, donor-site morbidity and risk of disease transmission, respectively, alternatives to traditional bone grafting options are warranted. To this end, poly(lactide-co-glycolide) (PLAGA) and in situ-synthesized amorphous hydroxyapatite (HA) were used to construct three-dimensional microsphere-based composite scaffolds of varying HA content for bone regeneration. In the current study, the effect of adding amorphous HA to the PLAGA scaffolds on their physical characteristics and in vitro degradation mechanism was investigated. Porosimetry and uniaxial compression testing were used to analyze the internal structure and elastic modulus of the scaffolds, respectively. Additionally, gel permeation chromatography (GPC) was performed to assess the polymer molecular weight over the course of an 8-week degradation study. HA content (17% or 27%) of the composite scaffolds was found to increase scaffold pore volume from 33.86% for pure polymer scaffolds, to 40.49% or 46.29%, depending on the amount of incorporated HA. This increased pore volume provided the composite scaffolds with a greater surface area and a corresponding decrease in elastic modulus. Scaffold degradation studies conducted over 8 weeks showed PLAGA to degrade in a first-order mechanism, with the rate of polymer degradation for the 27% HA composite scaffold being significantly slower than that of the pure PLAGA scaffold (degradation constants of 0.0324 and 0.0232 week(-1), respectively). These results suggest that the addition of amorphous HA to PLAGA microspheres resulted in porous, bioactive scaffolds that offer potential as alternative bone grafting materials for the field of regenerative medicine.

Apatite nano-crystalline surface modification of poly(lactide-co-glycolide) sintered microsphere scaffolds for bone tissue engineering: implications for protein adsorption.

A number of bone tissue engineering approaches are aimed at (i) increasing the osteconductivity and osteoinductivity of matrices, and (ii) incorporating bioactive molecules within the scaffolds. In this study we examined the growth of a nano-crystalline mineral layer on poly(lactide-co-glycolide) (PLAGA) sintered microsphere scaffolds for tissue engineering. In addition, the influence of the mineral precipitate layer on protein adsorption on the scaffolds was studied. Scaffolds were mineralized by incubation in simulated body fluid (SBF). Scanning electron microscopy (SEM) analysis revealed that mineralized scaffolds possess a rough surface with a plate-like nanostructure covering the surface of microspheres. The results of protein adsorption and release studies showed that while the protein release pattern was similar for PLAGA and mineralized PLAGA scaffolds, precipitation of the mineral layer on PLAGA led to enhanced protein adsorption and slower protein release. Mineralization of tissue-engineered surfaces provides a method for both imparting bioactivity and controlling levels of protein adsorption and release.

Human endothelial cell growth and phenotypic expression on three dimensional poly(lactide-co-glycolide) sintered microsphere scaffolds for bone tissue engineering.

Bone tissue engineering offers promising alternatives to repair and restore tissues. Our laboratory has employed poly(lactide-co-glycolide) PLAGA microspheres to develop a three dimensional (3-D) porous bioresorbable scaffold with a biomimetic pore structure. Osseous healing and integration with the surrounding tissue depends in part on new blood vessel formation within the porous structure. Since endothelial cells play a key role in angiogenesis (formation of new blood vessels from pre-existing vasculature), the purpose of this study was to better understand human endothelial cell attachment, viability, growth, and phenotypic expression on sintered PLAGA microsphere scaffold. Scanning electron microscopy (SEM) examination showed cells attaching to the surface of microspheres and bridging the pores between the microspheres. Cell proliferation studies indicated that cell number increased during early stages and reached a plateau between days 10 and 14. Immunofluorescent staining for actin showed that cells were proliferating three dimensionally through the scaffolds while staining for PECAM-1 (platelet endothelial cell adhesion molecule) displayed typical localization at cell-cell contacts. Gene expression analysis showed that endothelial cells grown on PLAGA scaffolds maintained their normal characteristic phenotype. The cell proliferation and phenotypic expression were independent of scaffold pore architecture. These results demonstrate that PLAGA sintered microsphere scaffolds can support the growth and biological functions of human endothelial cells. The insights from this study should aid future studies aimed at enhancing angiogenesis in three dimensional tissue engineered scaffolds.

Novel polymer-synthesized ceramic composite-based system for bone repair: an in vitro evaluation.

The emergence of synthetic bone repair scaffolds has been necessitated by the limitations of both autografts and allografts. Several candidate materials are available including degradable polymers and ceramics. However, these materials possess their own limitations that at least in part may be overcome by combining the two materials into a composite. Toward that end, a novel approach to forming a polymer/ceramic composite has been developed that combines degradable poly(lactide-co-glycolide) microspheres and a poorly crystalline calcium phosphate that is synthesized within the microspheres, which are then fused together to form a porous three-dimensional scaffold for bone repair. The design, fabrication, and characterization of the composite microspheres, the calcium phosphate formed within these microspheres, and the formation of scaffolds were studied. The calcium phosphate formed was analyzed by x-ray diffraction, Fourier transform infrared spectroscopy, and energy dispersive spectroscopy, and was shown to be similar to native bone in both composition and crystallinity by controlling certain processing parameters such as mixing time, solution pH, and mixing temperature. Scaffolds with porous interconnected structures and mechanical properties in the range of trabecular bone were fabricated via precise control of polymer/ceramic ratios within the microspheres and scaffold heating times. This composite scaffold represents a new and important vehicle for bone-tissue engineering.