Angela S.P. Lin

Microarchitectural and mechanical characterization of oriented porous polymer scaffolds.

Biodegradable porous polymer scaffolds are widely used in tissue engineering to provide a structural template for cell seeding and extracellular matrix formation. Scaffolds must often possess sufficient structural integrity to temporarily withstand functional loading in vivo or cell traction forces in vitro. Both the mechanical and biological properties of porous scaffolds are determined in part by the local microarchitecture. Quantification of scaffold structure-function relationships is therefore critical for optimizing mechanical and biological performance. In this study, porous poly(L-lactide-co-DL-lactide) scaffolds with axially oriented macroporosity and random microporosity were produced using a solution coating and porogen decomposition method. Microarchitectural parameters were quantified as a function of porogen concentration using microcomputed tomography (micro-CT) analysis and related to compressive mechanical properties. With increasing porogen concentration, volume fraction decreased consistently due to microarchitectural changes in average strut thickness, spacing, and density. The three-dimensional interconnectivity of the scaffold porosity was greater than 99% for all porogen concentration levels tested. Over a porosity range of 58-80%, the average compressive modulus and ultimate strength of the scaffolds ranged from 43.5-168.3 MPa and 2.7-11.0 MPa, respectively. Thus, biodegradable porous polymer scaffolds have been produced with oriented microarchitectural features designed to facilitate vascular invasion and cellular attachment and with initial mechanical properties comparable to those of trabecular bone.

Periosteal progenitor cell fate in segmental cortical bone graft transplantations: implications for functional tissue engineering.

A murine segmental femoral bone graft model was used to show the essential role of donor periosteal progenitor cells in bone graft healing. Transplantation of live bone graft harvested from Rosa 26A mice showed that ∼70% of osteogenesis on the graft was attributed to the expansion and differentiation of donor periosteal progenitor cells. Furthermore, engraftment of BMP‐2‐producing bone marrow stromal cells on nonvital allografts showed marked increases in cortical graft incorporation and neovascularization, suggesting that gene‐enhanced, tissue engineered functional periosteum may improve allograft incorporation and repair.

Hyaluronic acid hydrogels with controlled degradation properties for oriented bone regeneration

Non-healing fractures can result from trauma, disease, or age-related bone loss. While many treatments focus on restoring bone volume, few try to recapitulate bone organization. However, the native architecture of bone is optimized to provide its necessary mechanical properties. Hyaluronic acid (HA) hydrogel scaffold systems with tunable degradation properties were developed for the controlled delivery of osteoinductive and angiogenic growth factors, thus affecting the quantity and quality of regenerated tissue. HA hydrogels were designed to degrade at fast, intermediate, and slow rates due to hydrolysis and further provided controlled release of cationic proteins due to electrostatic interactions. Scaffolds delivering bone morphogenetic protein-2 (BMP-2) were evaluated in a rat calvarial bone critical size defect model. BMP-2 delivery from the HA hydrogels had a clear osteoinductive effect in vivo and, for all hydrogel types, BMP-2 delivery resulted in significant mineralization compared to control hydrogels. The temporal progression of this effect could be modulated by altering the degradation rate of the scaffold. All three degradation rates tested resulted in similar amounts of mineral formation at the latest (six week) time point examined. Interestingly, however, the fastest and slowest degrading scaffolds seemed to result in more organized bone than the intermediate degrading scaffold, which was designed to degrade in 6-8 weeks to match the healing time. Additionally, healing could be enhanced by co-delivery of vascular endothelial growth factor along with BMP-2.

Quantitative Assessment of Articular Cartilage Morphology via EPIC-μCT

OBJECTIVE The objective of the present study was to validate the ability of Equilibrium Partitioning of an Ionic Contrast agent via microcomputed tomography (EPIC-microCT) to nondestructively assess cartilage morphology in the rat model. DESIGN An appropriate contrast agent (Hexabrix) concentration and incubation time for equilibration were determined for reproducible segmentation of femoral articular cartilage from contrast-enhanced microCT scans. Reproducibility was evaluated by triplicate scans of six femora, and the measured articular cartilage thickness was independently compared to thickness determined from needle probe testing and histology. The validated technique was then applied to quantify age-related differences in articular cartilage morphology between 4, 8, and 16-week-old (n=5 each) male Wistar rats. RESULTS A 40% Hexabrix/60% phosphate buffered saline (PBS) solution with 30 min incubation was optimal for segmenting cartilage from the underlying bone tissue and other soft tissues in the rat model. High reproducibility was indicated by the low coefficient of variation (1.7-2.5%) in cartilage volume, thickness and surface area. EPIC-microCT evaluation of thickness showed a strong linear relationship and good agreement with both needle probing (r(2)=0.95, slope=0.81, P<0.01, mean difference 11+/-22 microm, n=43) and histology (r(2)=0.99, slope=0.97, P<0.01, mean difference 12+/-10 microm, n=30). Cartilage volume and thickness significantly decreased with age while surface area significantly increased. CONCLUSION EPIC-microCT imaging has the ability to nondestructively evaluate three-dimensional articular cartilage morphology with high precision and accuracy in a small animal model.

3D imaging of tissue integration with porous biomaterials

Porous biomaterials designed to support cellular infiltration and tissue formation play a critical role in implant fixation and engineered tissue repair. The purpose of this Leading Opinion Paper is to advocate the use of high resolution 3D imaging techniques as a tool to quantify extracellular matrix formation and vascular ingrowth within porous biomaterials and objectively compare different strategies for functional tissue regeneration. An initial over-reliance on qualitative evaluation methods may have contributed to the false perception that developing effective tissue engineering technologies would be relatively straightforward. Moreover, the lack of comparative studies with quantitative metrics in challenging pre-clinical models has made it difficult to determine which of the many available strategies to invest in or use clinically for companies and clinicians, respectively. This paper will specifically illustrate the use of microcomputed tomography (micro-CT) imaging with and without contrast agents to nondestructively quantify the formation of bone, cartilage, and vasculature within porous biomaterials.

Nondestructive Assessment of sGAG Content and Distribution in Normal and Degraded Rat Articular Cartilage via EPIC-μCT

OBJECTIVE The objective of this study was to evaluate the feasibility of quantifying the Equilibrium Partitioning of an Ionic Contrast agent via Microcomputed Tomography (EPIC-microCT) to nondestructively assess sulfated glycosaminoglycan (sGAG) content and distribution in rat articular cartilage ex vivo, and in doing so to establish a paradigm for extension of this technique to other small animal models. DESIGN After determination of an appropriate incubation time for the anionic contrast agent, EPIC-microCT was used to examine age-related differences in cartilage sGAG content between 4-, 8-, and 16-week old (n=5 each) male Wistar rats and to evaluate sGAG depletion in the right femora of each age group after 60 min of digestion with chondroitinase ABC. The EPIC-microCT measurements were validated by histological safranin-O staining, and reproducibility was evaluated by triplicate scans of six femora. RESULTS Cartilage attenuation gradually increased with cumulative digestion time and reached a plateau at approximately 60 min with a 16.0% temporal increase (P<0.01). Average femoral articular cartilage attenuation increased by 14.2% from 4- to 8-weeks of age (P<0.01) and further increased by 2.5% from 8 to 16 weeks (P<0.05). After 60 min of digestion, femoral articular cartilage attenuations increased by 15-17% in each age group (P<0.01). Correspondingly, sGAG optical density decreased with age and digestion, and showed a linear correlation (r=-0.88, slope=-1.26, P<0.01, n=30) with EPIC-microCT cartilage attenuation. High reproducibility was indicated by a low coefficient of variation (1.5%) in cartilage attenuation. CONCLUSIONS EPIC-microCT imaging provides high spatial resolution and sensitivity to assess sGAG content and three-dimensional distribution in rat femoral articular cartilage.

Rescue of Impaired Fracture Healing in COX-2−/− Mice via Activation of Prostaglandin E2 Receptor Subtype 4

Although the essential role of cyclooxygenase (COX)-2 in fracture healing is known, the targeted genes and molecular pathways remain unclear. Using prostaglandin E2 receptor (EP)2 and EP4 agonists, we examined the effects of EP receptor activation in compensation for the lack of COX-2 during fracture healing. In a fracture-healing model, COX-2(-/-) mice showed delayed initiation and impaired endochondral bone repair, accompanied by a severe angiogenesis deficiency. The EP4 agonist markedly improved the impaired healing in COX-2(-/-) mice, as evidenced by restoration of bony callus formation on day 14, a near complete reversal of bone formation, and an approximately 70% improvement of angiogenesis in the COX-2(-/-) callus. In comparison, the EP2 agonist only marginally enhanced bone formation in COX-2(-/-) mice. To determine the differential roles of EP2 and EP4 receptors on COX-2-mediated fracture repair, the effects of selective EP agonists on chondrogenesis were examined in E11.5 long-term limb bud micromass cultures. Only the EP4 agonist significantly increased cartilage nodule formation similar to that observed during prostaglandin E2 treatment. The prostaglandin E2/EP4 agonist also stimulated MMP-9 expression in bone marrow stromal cell cultures. The EP4 agonist further restored the reduction of MMP-9 expression in the COX-2(-/-) fracture callus. Taken together, our studies demonstrate that EP2 and EP4 have differential functions during endochondral bone repair. Activation of EP4, but not EP2 rescued impaired bone fracture healing in COX-2(-/-) mice.

Analyzing bone, blood vessels, and biomaterials with microcomputed tomography

Quantitative tools such as micro-CT are needed for tissue engineering to evolve beyond a qualitative, observational field and accelerate the clinical realization of regenerative technologies. As faster, higher-resolution micro-CT systems become available for both in vitro and in vivo studies and the development of improved contrast agents allows micro-CT imaging to be extended to nonmineralized tissues, additional novel applications related to tissue engineering are sure to emerge.

Convergence in a mechanically complex phenotype: detecting structural adaptations for crushing in cichlid fish.

Abstract Morphological convergence provides strong evidence that evolution is adaptive. However, putatively convergent morphology is often examined in two dimensions with no explicit model of function. In this study, we investigated structural and mechanical similarities of the lower pharyngeal jaw (LPJ) in cichlid fish that have evolved the ability to crush hard-shelled molluscs. Using a novel phylogeny, we demonstrated molluscivory has been gained and/or been lost numerous times in Heroine cichlids. Within this comparative framework, we produced three-dimensional computed tomography (CT) scans for LPJs of both morphotypes in the trophically polymorphic Herichthys minckleyi and six evolutionarily independent pairs of closely related species. Like H. minckleyi, these species exhibit divergence between a molluscivore and a nonmolluscivore. Using the CT scans, we generated finite element models of papilliform H. minckleyi LPJs to determine where stress would concentrate in a jaw not modified to crush molluscs. Then, we examined whether stress in the papilliform LPJ predicted structural modifications in molariform H. minckleyi and other molluscivorous species. Despite potential constraints, stresses imposed during prey processing explain 40% of LPJ variation in mollusc crushing species. The structural and mechanical analyses also suggest divergence found in polymorphic species could provide the substrate for trophic differences found in reproductively isolated cichlids.

Effects of In Vivo Mechanical Loading on Large Bone Defect Regeneration

Fracture healing is highly sensitive to mechanical conditions; however, the effects of mechanical loading on large bone defect regeneration have not been evaluated. In this study, we investigated the effects of functional loading on repair of critically sized segmental bone defects. About 6‐mm defects were created in rat femora, and each defect received 5 µg recombinant human bone morphogenetic protein‐2 (rhBMP‐2), delivered in alginate hydrogel. Limbs were stabilized by either stiff fixation plates for the duration of the study or compliant plates that allowed transfer of compressive ambulatory loads beginning at week 4. Healing was assessed by digital radiography, microcomputed tomography, mechanical testing, histology, and finite element modeling. Loading significantly increased regenerate bone volume and average polar moment of inertia. The response to loading was location‐dependent with the polar moment of inertia increased at the proximal end of the defect but not the distal end. As a result, torsional stiffness was 58% higher in the compliant plate group, but failure torque was not altered. In single samples assessed for histology from each group, a qualitatively greater amount of cartilage and a lesser degree of remodeling to lamellar bone occurred in the loaded group compared to the stiff plate group. Finally, principal strain histograms, calculated by FE modeling, revealed that the compliant plate samples had adapted to more efficiently distribute loads in the defects. Together, these data demonstrate that functional transfer of axial loads alters BMP‐induced large bone defect repair by increasing the amount and distribution of bone formed within the defect.