Evaluation of multi-scale mineralized collagen-polycaprolactone composites for bone tissue engineering.

A particular challenge in biomaterial development for treating orthopedic injuries stems from the need to balance bioactive design criteria with the mechanical and geometric constraints governed by the physiological wound environment. Such trade-offs are of particular importance in large craniofacial bone defects which arise from both acute trauma and chronic conditions. Ongoing efforts in our laboratory have demonstrated a mineralized collagen biomaterial that can promote human mesenchymal stem cell osteogenesis in the absence of osteogenic media but that possesses suboptimal mechanical properties in regards to use in loaded wound sites. Here we demonstrate a multi-scale composite consisting of a highly bioactive mineralized collagen-glycosaminoglycan scaffold with micron-scale porosity and a polycaprolactone support frame (PCL) with millimeter-scale porosity. Fabrication of the composite was performed by impregnating the PCL support frame with the mineral scaffold precursor suspension prior to lyophilization. Here we evaluate the mechanical properties, permeability, and bioactivity of the resulting composite. Results indicated that the PCL support frame dominates the bulk mechanical response of the composite resulting in a 6000-fold increase in modulus compared to the mineral scaffold alone. Similarly, the incorporation of the mineral scaffold matrix into the composite resulted in a higher specific surface area compared to the PCL frame alone. The increased specific surface area in the collagen-PCL composite promoted increased initial attachment of porcine adipose derived stem cells versus the PCL construct.

Mandibular reconstruction with a bioactive-coated cementless Ti6Al4V modular endoprosthesis in Macaca fascicularis.

The titanium mandibular modular endoprosthesis fixed with polymethylmethacrylate cement in the medullary space of the mandible has been introduced in previous studies. However, the internal parts of these devices have been found to be prone to loosening and wound dehiscence. The current study introduces a newly designed bioactive-coated cementless modular mandibular endoprosthesis, which was used for reconstruction in Macaca fascicularis. The devices were coated with hydroxyapatite (HA) and hydroxyapatite/bioglass (HA/BG) and implanted in unilateral mandibular segmental defects in nine monkeys for 6 months. Biomechanical testing found the reconstructed mandible to have a mean stiffness value of 110.43 N/mm. Histologically, there were both fibrous capsule and woven bone around the device body, and histomorphology analysis showed 64.17% bone contact to device stem surface. The percentage bone volume calculated from micro-computed tomography analysis around the stem surface was found to be superior to that reported in previous studies of cemented mandibular endoprostheses. Intermodular connection screw loosening has also been resolved with the dovetail interconnection. In conclusion, the current bioactive-coated cementless mandibular endoprosthesis is feasible for use in mandibular segmental reconstruction. However, insufficient load-bearing capability and a high rate of intraoral wound dehiscence were found in the majority of the study animals. Further device modifications and improvements in the surgical technique need to be addressed in future studies.

Permeability analysis of scaffolds for bone tissue engineering.

Porous artificial bone substitutes, especially bone scaffolds coupled with osteobiologics, have been developed as an alternative to the traditional bone grafts. The bone scaffold should have a set of properties to provide mechanical support and simultaneously promote tissue regeneration. Among these properties, scaffold permeability is a determinant factor as it plays a major role in the ability for cells to penetrate the porous media and for nutrients to diffuse. Thus, the aim of this work is to characterize the permeability of the scaffold microstructure, using both computational and experimental methods. Computationally, permeability was estimated by homogenization methods applied to the problem of a fluid flow through a porous media. These homogenized permeability properties are compared with those obtained experimentally. For this purpose a simple experimental setup was used to test scaffolds built using Solid Free Form techniques. The obtained results show a linear correlation between the computational and the experimental permeability. Also, this study showed that permeability encompasses the influence of both porosity and pore size on mass transport, thus indicating its importance as a design parameter. This work indicates that the mathematical approach used to determine permeability may be useful as a scaffold design tool.

Strategies for regeneration of the bone using porcine adult adipose-derived mesenchymal stem cells.

Bone is a plastic tissue with a large healing capability. However, extensive bone loss due to disease or trauma requires tissue-engineering applications. Presently, bone grafting is the gold standard for bone repair, but presents serious limitations including donor site morbidity, rejection, and limited tissue regeneration. The use of stem cells appears to be a means to overcome such limitations. Bone marrow mesenchymal stem cells (BMSC) have been the choice, thus far, for stem cell therapy for bone regeneration. However, it has been shown that adipose-derived stem cells (ASC) have similar immunophenotype, morphology, multilineage potential, and transcriptome compared to BMSC. Moreover, ASC are much more abundant, more accessible and have lower donor morbidity, which combined may make ASC a better alternative to BMSC. ASC are also able to migrate to the site of injury and have immunosuppressive abilities similar to BMSC. Further, ASC have demonstrated extensive osteogenic capacity both in vitro and in vivo in several species, greatly enhancing the healing of critical size defects. The use of scaffolds in combination with ASC and growth factors provides a valuable tool for guided bone regeneration, especially for complex anatomic defects. Some critical elements include ASC-scaffold interactions and appropriate three-dimensional design of the porous osteoinductive structures. This review examines data that provides strong support for the clinical translation of ASC for bone regeneration.

Computed tomography-based tissue-engineered scaffolds in craniomaxillofacial surgery.

INTRODUCTION: Tissue engineering provides an alternative modality allowing for decreased morbidity of donor site grafting and decreased rejection of less compatible alloplastic tissues. METHODS: Using image-based design and computer software, a precisely sized and shaped scaffold for osseous tissue regeneration can be created via selective laser sintering. Polycaprolactone has been used to create a condylar ramus unit (CRU) scaffold for application in temporomandibular joint reconstruction in a Yucatan minipig animal model. Following sacrifice, micro-computed tomography and histology was used to demonstrate the efficacy of this particular scaffold design. RESULTS: A proof-of-concept surgery has demonstrated cartilaginous tissue regeneration along the articulating surface with exuberant osseous tissue formation. Bone volumes and tissue mineral density at both the 1 and 3 month time points demonstrated significant new bone growth interior and exterior to the scaffold. CONCLUSION: Computationally designed scaffolds can support masticatory function in a large animal model as well as both osseous and cartilage regeneration. Our group is continuing to evaluate multiple implant designs in both young and mature Yucatan minipig animals.

Localized viral vector delivery to enhance in situ regenerative gene therapy.

A lyophilization method was developed to locally release adenoviral vectors directly from biomaterials for in situ regenerative gene therapy. Adenovirus expressing a beta-galactosidase reporter gene (AdLacZ) was mixed with different excipient formulations and lyophilized on hydroxyapatite (HA) disks followed by fibroblasts culturing and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) staining, suggesting 1 M sucrose in phosphate-buffered saline had best viability. Adenovirus release studies showed that greater than 30% virus remained on the material surface up to 16 h. Lyophilized adenovirus could be precisely localized in defined patterns and the transduction efficiency was also improved. To determine if the lyophilization formulations could preserve viral bioactivity, the lyophilized AdLacZ was tested after being stored at varying temperatures. Bioactivity of adenovirus lyophilized on HA was maintained for greater than 6 months when stored at -80 degrees C. In vivo studies were performed using an adenovirus encoding BMP-2 (AdBMP-2). AdBMP-2 was lyophilized in gelatin sponges and placed into rat critical-size calvarial defects for 5 weeks. Micro-computed tomography (micro-CT) analysis demonstrated that free-form delivery of AdBMP-2 had only modest effects on bone formation. In contrast, AdBMP-2 lyophilized in gelatin sponges led to more than 80% regeneration of critical-size calvarial defects.

Craniofacial tissue engineering by stem cells.

Craniofacial tissue engineering promises the regeneration or de novo formation of dental, oral, and craniofacial structures lost to congenital anomalies, trauma, and diseases. Virtually all craniofacial structures are derivatives of mesenchymal cells. Mesenchymal stem cells are the offspring of mesenchymal cells following asymmetrical division, and reside in various craniofacial structures in the adult. Cells with characteristics of adult stem cells have been isolated from the dental pulp, the deciduous tooth, and the periodontium. Several craniofacial structures--such as the mandibular condyle, calvarial bone, cranial suture, and subcutaneous adipose tissue--have been engineered from mesenchymal stem cells, growth factor, and/or gene therapy approaches. As a departure from the reliance of current clinical practice on durable materials such as amalgam, composites, and metallic alloys, biological therapies utilize mesenchymal stem cells, delivered or internally recruited, to generate craniofacial structures in temporary scaffolding biomaterials. Craniofacial tissue engineering is likely to be realized in the foreseeable future, and represents an opportunity that dentistry cannot afford to miss.

Tissue engineering osteochondral implants for temporomandibular joint repair.

Tissue engineering has provided an alternative to traditional strategies to repair and regenerate temporomandibular joints (TMJ). A successful strategy to engineer osteochondral tissue, such as that found in the TMJ, will produce tissue that is both biologically and mechanically functional. Image-based design (IBD) and solid free-form (SFF) fabrication can be used to generate scaffolds that are load bearing and match patient and defect site geometry. The objective of this study was to demonstrate how scaffold design, materials, and biological factors can be used in an integrated approach to regenerate a multi-tissue interface. IBD and SFF were first used to create biomimetic scaffolds with appropriate bulk geometry and microarchitecture. Biphasic composite scaffolds were then manufactured with the same techniques and used to simultaneously generate bone and cartilage in discrete regions and provide for the development of a stable interface between cartilage and subchondral bone. Poly-l-lactic acid/hydroxyapatite composite scaffolds were differentially seeded with fibroblasts transduced with an adenovirus expressing bone morphogenetic protein-7 in the ceramic phase and fully differentiated chondrocytes in the polymeric phase, and were subcutaneously implanted into mice. Following implantation in the ectopic site, the biphasic scaffolds promoted the simultaneous growth of bone, cartilage, and a mineralized interface tissue. Within the ceramic phase, the pockets of tissue generated included blood vessels, marrow stroma, and adipose tissue. This combination of IBD and SFF-fabricated biphasic scaffolds with gene and cell therapy is a promising approach to regenerate osteochondral defects and, ultimately, the TMJ.

Engineering craniofacial scaffolds.

OBJECTIVE: To develop an integrated approach for engineering craniofacial scaffolds and to demonstrate that these engineered scaffolds would have mechanical properties in the range of craniofacial tissue and support bone regeneration for craniofacial reconstruction. EXPERIMENTAL VARIABLE: Scaffold architecture designed to achieve desired elasticity and permeability. Scaffold external shape designed to match craniofacial anatomy. OUTCOME MEASURE: Final fabricated biomaterial scaffolds. Compressive mechanical modulus and strength. Bone regeneration as measured by micro-CT scanning, mechanical testing and histology. SETTING: Departments of Biomedical Engineering, Oral/Maxillofacial Surgery, and Oral Medicine, Pathology and Oncology at the University of Michigan. RESULTS: Results showed that the design/fabrication approach could create scaffolds with designed porous architecture to match craniofacial anatomy. These scaffolds could be fabricated from a wide range of biomaterials, including titanium, degradable polymers, and degradable calcium phosphate ceramics. Mechanical tests showed that fabricated scaffolds had compressive modulus ranging 50 to 2900 MPa and compressive strength ranging from 2 to over 56 MPa, within the range of human craniofacial trabecular bone. In vivo testing of designed scaffolds showed that they could support bone regeneration via delivery of BMP-7 transduced human gingival fibroblasts in a mouse model. Designed hydroxyapatite scaffolds with pore diameters ranging from 400 to 1200 microns were implanted in minipig mandibular defects for 6 and 18 weeks. Results showed substantial bone ingrowth (between 40 and 50% at 6 weeks, between 70 and 80% at 18 weeks) for all scaffolds, with no significant difference based on pore diameter. CONCLUSION: Integrated image-based design and solid free-form fabrication can create scaffolds that attain desired elasticity and permeability while fitting any 3D craniofacial defect. The scaffolds could be manufactured from degradable polymers, calcium phosphate ceramics and titanium. The designed scaffolds supported significant bone regeneration for all pore sizes ranging from 300 to 1200 microns. These results suggest that designed scaffolds are clinically applicable for complex craniofacial reconstruction.

Inclusion of organ deformation in dose calculations.

A previously described system for modeling organ deformation using finite element analysis has been extended to permit dose calculation. Using this tool, the calculated dose to the liver during radiotherapy can be compared using a traditional static model (STATIC), a model including rigid body motion (RB), and finally a model that incorporates rigid body motion and deformation (RBD). A model of the liver, consisting of approximately 6000 tetrahedral finite elements distributed throughout the contoured volume, is created from the CT data obtained at exhale. A deformation map is then created to relate the liver in the exhale CT data to the liver in the inhale CT data. Six intermediate phase positions of each element are then calculated from their trajectories. The coordinates of the centroid of each element at each phase are used to determine the dose received. These intermediate dose values are then time weighted according to a population-modeled breathing pattern to determine the total dose to each element during treatment. This method has been tested on four patient datasets. The change in prescribed dose for each patient's actual tumor as well as a simulated tumor of the same size, located in the superior, intermediate, and inferior regions of the liver, was determined using a normal tissue complication model, maintaining a predicted probability of complications of 15%. The average change in prescribed dose from RBD to STATIC for simulated tumors in the superior, intermediate, and inferior regions are 4.0 (range 2.1 to 5.3), -3.6 (range -5.0 to -2.2), and -14.5 (range -27.0 to -10.0) Gy, respectively. The average change in prescribed dose for the patient's actual tumor was -0.4 Gy (range -4.1 to 1.7 Gy). The average change in prescribed dose from RBD to RB for simulated tumors in the superior, intermediate, and inferior regions are -0.04 (range -2.4 to 2.2), 0.2 (range -1.5 to 1.9), and 3.9 (range 0.8 to 7.3) Gy, respectively. The average change in the prescribed dose for the patient's actual tumor was 0.7 Gy (range 0.2 to 1.1 Gy). This patient sampling indicates the potential importance of including deformation in dose calculations.

Indirect solid free form fabrication of local and global porous, biomimetic and composite 3D polymer-ceramic scaffolds.

Precise control over scaffold material, porosity, and internal pore architecture is essential for tissue engineering. By coupling solid free form (SFF) manufacturing with conventional sponge scaffold fabrication procedures, we have developed methods for casting scaffolds that contain designed and controlled locally porous and globally porous internal architectures. These methods are compatible with numerous bioresorbable and non-resorbable polymers, ceramics, and biologic materials. Phase separation, emulsion-solvent diffusion, and porogen leaching were used to create poly(L)lactide (PLA) scaffolds containing both computationally designed global pores (500, 600, or 800 microm wide channels) and solvent fashioned local pores (50-100 microm wide voids or 5-10 microm length plates). Globally porous PLA and polyglycolide/PLA discrete composites were made using melt processing. Biphasic scaffolds with mechanically interdigitated PLA and sintered hydroxyapatite regions were fabricated with 500 and 600 microm wide global pores. PLA scaffolds with complex internal architectures that mimicked human trabecular bone were produced. Our indirect fabrication using casting in SFF molds provided enhanced control over scaffold shape, material, porosity and pore architecture, including size, geometry, orientation, branching, and interconnectivity. These scaffolds that contain concurrent local and global pores, discrete material regions, and biomimetic internal architectures may prove valuable for multi-tissue and structural tissue interface engineering.

Technical note: creating a four-dimensional model of the liver using finite element analysis.

Finite element analysis and two liver CT scans were used to construct a four-dimensional (4D) model of the liver during breathing. A linear elastic, small deformation mechanical model was applied to one patient to obtain intermediate organ position and shape between exhale and inhale. Known transformations between anatomically defined subsections of the exhale and inhale liver surfaces were applied as constraints to the exhale CT liver model. Intermediate states were then calculated and time weighted to determine a 4D model of the liver as it deforms during the breathing cycle. This model can be used to calculate a more accurate dose distribution during radiotherapy.

Optimal design and fabrication of scaffolds to mimic tissue properties and satisfy biological constraints.

Bone tissue engineering scaffolds must shape regenerating tissue, provide temporary mechanical support and enhance tissue regeneration. These requirements result in conflicting design goals. For example, increased temporary mechanical function requires a dense scaffold while enhanced cell/gene delivery requires a porous scaffold. This paper demonstrates an image-based homogenization optimization approach that can design scaffold microstructure, scaffold material and regenerate tissue microstructure to meet conflicting design requirements. In addition, constraints to ensure adequate cell/gene delivery can be introduced using a minimum porosity threshold. Homogenization theory was used to compute relationships between scaffold microstructure and effective stiffness. The functional relationships were used in the MATLAB optimization toolbox to compute optimal pore dimensions and scaffold material such that the scaffold and regenerate tissue effective stiffness matched that of native bone stiffness. The scaffold design was converted into STL format for solid free-form fabrication. Scaffolds were designed that matched mandibular condyle trabecular bone properties. Results showed excellent agreement between native bone properties and designed scaffold properties (all R2 > 0.89). Finally, example scaffolds were built from hydroxyapatite using a SFF casting technique.

Manufacturing and characterization of 3-d hydroxyapatite bone tissue engineering scaffolds.

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.

Trabecular surface remodeling simulation for cancellous bone using microstructural voxel finite element models.

A computational simulation method for three-dimensional trabecular surface remodeling was proposed, using voxel finite element models of cancellous bone, and was applied to the experimental data. In the simulation, the trabecular microstructure was modeled based on digital images, and its morphological changes due to surface movement at the trabecular level were directly expressed by removing/adding the voxel elements from/to the trabecular surface. A remodeling simulation at the single trabecular level under uniaxial compressive loading demonstrated smooth morphological changes even though the trabeculae were modeled with discrete voxel elements. Moreover, the trabecular axis rotated toward the loading direction with increasing stiffness, simulating functional adaptation to the applied load. In the remodeling simulation at the trabecular structural level, a cancellous bone cube was modeled using a digital image obtained by microcomputed tomography (microCT), and was uniaxially compressed. As a result, the apparent stiffness against the applied load increased by remodeling, in which the trabeculae reoriented to the loading direction. In addition, changes in the structural indices of the trabecular architecture coincided qualitatively with previously published experimental observations. Through these studies, it was demonstrated that the newly proposed voxel simulation technique enables us to simulate the trabecular surface remodeling and to compare the results obtained using this technique with the in vivo experimental data in the investigation of the adaptive bone remodeling phenomenon.

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.

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 degrees 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 microm and 968 microm in diameter with standard deviations of 50 microm 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 controlled channel size can be built with the technique developed in this study.

An image-based approach for designing and manufacturing craniofacial scaffolds.

Bone tissue engineering (BTE), which combines biomaterial scaffolds with biologically active factors, holds tremendous promise for reconstructing craniofacial defects. A significant challenge in craniofacial reconstructive BTE applications is the complex patient-specific geometry that must be reconstructed. In this paper, we present an image-based approach for designing and manufacturing patient-specific craniofacial biomaterial scaffolds directly from CT or MRI data. In this approach, voxel density distribution is used to define scaffold topology. The scaffold design topology is created using image processing techniques. This voxel density distribution is then converted to data that can be used to drive a Solid Free-Form Fabrication machine to either directly build the scaffold or build a mold for the scaffold. Several preliminary applications for craniofacial surgery, including a mandibular condyle scaffold, an orbital floor scaffold, and a general mandibular defect scaffold, are illustrated. Finally, we show applications to in vivo models.