Catherine G. Ambrose

Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner

Bone is a complex highly structured mechanically active 3D tissue composed of cellular and matrix elements. The true biological environment of a bone cell is thus derived from a dynamic interaction between responsively active cells experiencing mechanical forces and a continuously changing 3D matrix architecture. To investigate this phenomenon in vitro, marrow stromal osteoblasts were cultured on 3D scaffolds under flow perfusion with different rates of flow for an extended period to permit osteoblast differentiation and significant matrix production and mineralization. With all flow conditions, mineralized matrix production was dramatically increased over statically cultured constructs with the total calcium content of the cultured scaffolds increasing with increasing flow rate. Flow perfusion induced de novo tissue modeling with the formation of pore-like structures in the scaffolds and enhanced the distribution of cells and matrix throughout the scaffolds. These results represent reporting of the long-term effects of fluid flow on primary differentiating osteoblasts and indicate that fluid flow has far-reaching effects on osteoblast differentiation and phenotypic expression in vitro. Flow perfusion culture permits the generation and study of a 3D, actively modeled, mineralized matrix and can therefore be a valuable tool for both bone biology and tissue engineering.

Bioabsorbable implants: review of clinical experience in orthopedic surgery.

Bioabsorbable implants are widely used in orthopedic surgery today and the worldwide market is expanding rapidly. Despite the popularity of these implants, reports of complications continue to appear in the literature. Although the complications rarely have an adverse affect on long-term outcomes, the reports are too numerous to be mere isolated incidents related to one specific implant. Complications have been reported with most of the commercially available implant materials with varying incidence rates and severities of reactions to the implants. The purpose of this review is to summarize the adverse events that have been reported in clinical trials of bioabsorbable implants in orthopedic surgery.

Excessive transforming growth factor-β signaling is a common mechanism in osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a heritable disorder, in both a dominant and recessive manner, of connective tissue characterized by brittle bones, fractures and extraskeletal manifestations. How structural mutations of type I collagen (dominant OI) or of its post-translational modification machinery (recessive OI) can cause abnormal quality and quantity of bone is poorly understood. Notably, the clinical overlap between dominant and recessive forms of OI suggests common molecular pathomechanisms. Here, we show that excessive transforming growth factor-β (TGF-β) signaling is a mechanism of OI in both recessive (Crtap−/−) and dominant (Col1a2tm1.1Mcbr) OI mouse models. In the skeleton, we find higher expression of TGF-β target genes, higher ratio of phosphorylated Smad2 to total Smad2 protein and higher in vivo Smad2 reporter activity. Moreover, the type I collagen of Crtap−/− mice shows reduced binding to the small leucine-rich proteoglycan decorin, a known regulator of TGF-β activity. Anti–TGF-β treatment using the neutralizing antibody 1D11 corrects the bone phenotype in both forms of OI and improves the lung abnormalities in Crtap−/− mice. Hence, altered TGF-β matrix-cell signaling is a primary mechanism in the pathogenesis of OI and could be a promising target for the treatment of OI.

In vitro degradation of polymeric networks of poly(propylene fumarate) and the crosslinking macromer poly(propylene fumarate)-diacrylate

Polymeric networks of poly(propylene fumarate) (PPF) crosslinked with poly(propylene fumarate)-diacrylate (PPF-DA) are currently being investigated as an injectable, biodegradable bone cement. This study examined the effect of crosslinking density, medium pH, and the incorporation of a beta-tricalcium phosphate (beta-TCP) filler on the in vitro degradation of PPF/PPF-DA. Cylindrical specimens were submerged in buffered saline at 37 degrees C and the change in weight, geometry, and compressive mechanical properties were monitored over a 52-week period. All formulations showed an initial increase in modulus and yield strength over the first 12 weeks, achieving maxima of 1307+/-101 and 51+/-3MPa, respectively, for the beta-TCP composite. PPF/PPF-DA networks with the lower crosslinking density demonstrated the greatest degradation with a 17% mass loss. Samples in the lower buffer pH 5.0 compared to physiological pH 7.4 did not show any differences in mass loss, but exhibited a faster decrease in the compressive strength over time. The beta-TCP composites maintained their mechanical properties at the level following their initial increase. These results show that the degradation of PPF/PPF-DA networks can be controlled by the crosslinking density, accelerated at a lower pH, and prolonged with the incorporation of the beta-TCP filler.

Flow perfusion culture of marrow stromal cells seeded on porous biphasic calcium phosphate ceramics

Calcium phosphate ceramics have been widely used for filling bone defects to aid in the regeneration of new bone tissue. Addition of osteogenic cells to porous ceramic scaffolds may accelerate the bone repair process. This study demonstrates the feasibility of culturing marrow stromal cells (MSCs) on porous biphasic calcium phosphate ceramic scaffolds in a flow perfusion bioreactor. The flow of medium through the scaffold porosity benefits cell differentiation by enhancing nutrient transport to the scaffold interior and by providing mechanical stimulation to cells in the form of fluid shear. Primary rat MSCs were seeded onto porous ceramic (60% hydroxyapatite, 40% β-tricalcium phosphate) scaffolds, cultured for up to 16 days in static or flow perfusion conditions, and assessed for osteoblastic differentiation. Cells were distributed throughout the entire scaffold by 16 days of flow perfusion culture whereas they were located only along the scaffold perimeter in static culture. At all culture times, flow perfused constructs demonstrated greater osteoblastic differentiation than statically cultured constructs as evidenced by alkaline phosphatase activity, osteopontin secretion into the culture medium, and histological evaluation. These results demonstrate the feasibility and benefit of culturing cell/ceramic constructs in a flow perfusion bioreactor for bone tissue engineering applications.

Effective treatment of osteomyelitis with biodegradable microspheres in a rabbit model.

Biodegradable microspheres were manufactured from a high molecular weight copolymer of 50% lactic and 50% glycolic acid and the antibiotic tobramycin. It was hypothesized that the microspheres would be more effective than polymethylmethacrylate beads in the local delivery of tobramycin and that the microspheres would not inhibit bone healing. Osteomyelitis was established in 40 New Zealand White rabbits using Staphylococcus aureus. All animals had irrigation and debridement of the infected radii four weeks after inoculation and were divided into five treatment groups: debridement alone, microspheres alone, microspheres containing tobramycin plus parenteral treatment with cefazolin, polymethylmethacrylate beads containing tobramycin plus parenteral cefazolin, and parenteral cefazolin. All animals were sacrificed after 4 weeks of treatment. The group treated with microspheres plus parenteral antibiotics was the only group to have a significantly higher percentage of animals without bacteria after 4 weeks of treatment when compared with the control group. Additionally, the animals treated with microspheres had a higher degree of bone healing in the defect than the animals treated with bone cement. The most effective treatment was biodegradable microspheres combined with parenteral antibiotic in this rabbit osteomyelitis model.

Antibiotic microspheres: Preliminary testing for potential treatment of osteomyelitis

Osteomyelitis is a difficult problem for orthopaedic surgeons. The current standard of treatment requires high doses of antibiotic to be administered parenterally, which can damage vital organs. A local drug delivery system, which targets only the infected tissues, would eliminate some of the complications associated with extended courses of parenteral antibiotic treatment. In the current study, biodegradable microspheres were manufactured from a high molecular weight copolymer of 50% lactic and 50% glycolic acid and the antibiotic tobramycin. Various formulations of microspheres were tested for in vitro elution characteristics to determine the optimum formulation for linear release of antibiotic for at least 4 weeks. The optimal formulation then was implanted into a pouch created in the quadriceps muscle of mice to evaluate the in vivo elution of the antibiotic and the inflammatory response elicited by the microspheres. Results indicate that a sustained linear release of antibiotic from the microspheres is possible for a period of at least 4 weeks and that the inflammatory response was within levels required for the microspheres to be considered biocompatible.

Fabrication of poly(propylene fumarate)-based orthopaedic implants by photo-crosslinking through transparent silicone molds

This work presents a new molding process for photo-crosslinked, degradable polymeric networks of poly(propylene fumarate) (PPF) and the crosslinking agent poly(propylene fumarate)-diacrylate (PPF-DA). Transparent room temperature vulcanizing silicone molds were fabricated for parts ranging from simple test coupons to orthopaedic implants. The PPF/PPF-DA resin blend was injected into the cavity and photo-crosslinked as light was transmitted through the mold wall. The volumetric shrinkage, mechanical properties, and the effects of gamma sterilization were reported for molded PPF/PPF-DA networks prepared with varying compositions of the two polymer components. The shrinkage decreased while the mechanical properties displayed a general increasing trend when more of the crosslinking agent was incorporated into the network. Gamma irradiation resulted in an improvement of the mechanical properties. In addition, PPF/PPF-DA replicates of a 70:30 poly(L/DL-lactide) biodegradable fixation plate and a bone allograft interbody fusion spacer were produced to evaluate the performance of PPF/PPF-DA as an orthopaedic implant and allow for a comparison to be made with materials that have been established for clinical use.

Tendon transfer fixation: Comparing a tendon to tendon technique vs. bioabsorbable interference-fit screw fixation

Tendon transfer techniques in the foot and ankle are used for tendon ruptures, deformities, and instabilities. This fresh cadaver study compares the tendon fixation strength in 10 paired specimens by performing a tendon to tendon fixation technique or using 7times20–25 mm bioabsorbable interference-fit screw tendon fixation technique. Load at failure of the tendon to tendon fixation method averaged 279N (Standard Deviation 81N) and the bioabsorbable screw 148N (Standard Deviation 72N) [p=0.0008]. Bioabsorbable interference-fit screws in these specimens show decreased fixation strength relative to the traditional fixation technique. However, the mean bioabsorbable screw fixation strength of 148N provides physiologic strength at the tendon-bone interface.

The swaying mouse as a model of osteogenesis imperfecta caused by WNT1 mutations

Osteogenesis imperfecta (OI) is a heritable disorder of connective tissue characterized by bone fragility and low bone mass. Recently, our group and others reported that WNT1 recessive mutations cause OI, whereas WNT1 heterozygous mutations cause early onset osteoporosis. These findings support the hypothesis that WNT1 is an important WNT ligand regulating bone formation and bone homeostasis. While these studies provided strong human genetic and in vitro functional data, an in vivo animal model to study the mechanism of WNT1 function in bone is lacking. Here, we show that Swaying (Wnt1(sw/sw)) mice previously reported to carry a spontaneous mutation in Wnt1 share major features of OI including propensity to fractures and severe osteopenia. In addition, biomechanical and biochemical analyses showed that Wnt1(sw/sw) mice exhibit reduced bone strength with altered levels of mineral and collagen in the bone matrix that is also distinct from the type I collagen-related form of OI. Further histomorphometric analyses and gene expression studies demonstrate that the bone phenotype is associated with defects in osteoblast activity and function. Our study thus provides in vivo evidence that WNT1 mutations contribute to bone fragility in OI patients and demonstrates that the Wnt1(sw/sw) mouse is a murine model of OI caused by WNT1 mutations.