Three-Dimensionally Printed Polycaprolactone and β-Tricalcium Phosphate Scaffolds for Bone Tissue Engineering: An In Vitro Study

Authors

Basel Sharaf, Caroline B. Faris, Harutsugi Abukawa, Srinivas M. Susarla, Joseph P. Vacanti, Leonard B. Kaban, Maria J. Troulis

Abstract

The purpose of this study was to evaluate porcine bone marrow–derived progenitor cell (pBMPC) proliferation and penetration into a novel 3-dimensionally printed scaffold. Four different tissue engineering scaffolds to evaluate pBMPC proliferation and penetration were examined. Scaffolds were fabricated from polycaprolactone (PCL) or the combination of β-tricalcium phosphate (β-TCP) and PCL (50:50), with 2 separate channel sizes (1 mm [small (S)] vs 2 mm [large (L)]). Scaffolds were fabricated into 20 × 20 × 7–mm blocks by use of a TheriForm machine (Integra Life Sciences, Akron, OH). Four groups of scaffolds were examined for pBMPC proliferation and penetration: group 1, β-TCP/PCL S; group 2, β-TCP/PCL L; group 3, PCL S; and group 4, PCL L. Nonparametric mean (Kruskal-Wallis) and multiple comparisons tests were used to compare the 4 groups. No shrinkage or deformation was noted in any of the scaffold groups after 2 weeks of culture. Mean surface cell counts ranged from 13.4 to 87.8 cells/0.57 mm2, with group 1 (β-TCP/PCL S) having statistically significantly higher counts than the other groups (P < .001). Mean interior cell counts ranged from 10.9 to 75.6 cells/0.57 mm2, with group 1 having the greatest interior cell count (P < .001). Total collagen formation ranged from 0.2% to 86%, with group 1 having the highest collagen formation (P < .001). The 3-dimensionally printed scaffold (β-TCP/PCL) with 1-mm channels showed greater cellular proliferation, penetration, and collagen formation after a 2-week in vitro culture than the other scaffolds evaluated. β-TCP/PCL S scaffolds warrant further evaluation for bone tissue engineering in vivo.

Link to Article

http://dx.doi.org/10.1016/j.joms.2011.07.029

Transgenic overexpression of bone morphogenetic protein 11 propeptide in skeleton enhances bone formation

Authors

Zicong Li, Fang Zeng, Alva Mitchell, Yong Soo Kim, Zhenfang Wu, Jinzeng Yang

Abstract

Bone morphogenetic protein 11 (BMP11) is a key regulatory protein in skeletal development. BMP11 propeptide has been shown to antagonize GDF11 activity in vitro. To explore the role of BMP11 propeptide in skeletal formation in vivo, we generated transgenic mice with skeleton-specific overexpression of BMP11 propeptide cDNA. The mice showed a transformation of the seventh cervical vertebra into a thoracic vertebra in our previous report. Presently, further characterizations of the transgenic mice indicated that ossification in calvatia was dramatically enhanced in transgenic fetuses at 16.5 dpc in comparison with their wild-type littermates. At 10 weeks of age, bone mineral content and bone mineral density were significantly (P < 0.05) higher in transgenic mice than that in their wild-type littermates based on dual energy X-ray absorptiometry analysis. The relative trabecular bone volume measured by histological analysis was dramatically increased in transgenic mice compared with their wild-type littermates. The enhanced bone formations in the transgenic mice appear to result from increase osteoblast activities as the expressions of four osteoblast markers – α1 type 1 collagen, osteocalcin, alkaline phosphatase and phex were significantly higher in transgenic fetuses than that in their wild-type littermates. These results suggest that over-expression of BMP11 propeptide stimulates bone formation by increasing osteoblast cell functions.

Link to Article

http://dx.doi.org/10.1016/j.bbrc.2011.11.019

Femoral metaphysis bending test of rat: introduction and validation of a novel biomechanical testing protocol for osteoporosis

Authors

BaiLing Chen, YiQiang Li, XiaoXi Yang and DengHui Xie

Abstract

The diaphysis bending test is generally accepted to assess the biomechanical properties of bone in osteoporotic animals. However, bone strength loss was more pronounced at the metaphysis than diaphysis. Therefore, the biomechanical test should be focused on the metaphysis. This study aimed to validate a novel biomechanical test for femoral metaphysis in ovariectomized rats. Twenty 5-month-old female Sprague-Dawley rats were randomly divided into the ovariectomized (OVX) and sham-operated (Sham) groups. Examination of femur bone mineral density (BMD) and histomorphometry of the distal femur were performed. Femur biomechanical parameters (maximal load, yield load, and stiffness) were determined by the diaphysis bending test and a novel designed metaphysis bending test. Pearson's correlations were used to analyze the relationships between the biomechanical parameters and BMD or bone histomorphometry indexes (%Tb.Ar, Tb.N, Tb.Th), respectively. The femur BMD, bone histomorphometry indexes, and biomechanical parameters of OVX were inferior to those of the Sham group (P < 0.05). In the diaphysis bending test, the mean difference of the maximum load and yield load between the OVX and Sham groups were 13.83 ± 5.27 and 15.69 ± 4.15 N, which were significantly lower than in the metaphysis bending test (43.34 ± 4.27, 48.90 ± 4.35 N; all P < 0.05). Positive correlations between biomechanical parameters and femur BMD or bone histomorphometry indexes were observed in both the diaphysis bending and metaphysis bending test. The biomechanical parameters in the metaphysis bending test showed stronger correlations with BMD and bone histomorphometry indexes. The femoral metaphysis bending test was validated to assess osteoporosis in our study, and it was more sensitive than the diaphysis bending test in evaluating the change of biomechanical properties of the femur in osteoporotic rats.

Link to Article

http://dx.doi.org/10.1007/s00776-011-0167-7

Structural characteristics of the collagen network in human normal, degraded and repair articular cartilages observed in polarized light and scanning electron microscopies

Authors

A. Changoor†, , M. Nelea†, S. Méthot‡, N. Tran-Khanh†, A. Chevrier†, A. Restrepo‡, M.S. Shive‡, C.D. Hoemann†, M.D. Buschmann

Abstract

This study characterizes collagen organization (CO) in human normal (n = 6), degraded (n = 6) and repair (n = 22) cartilages, using polarized light (PLM) and scanning electron (SEM) microscopies. CO was assessed using a recently developed PLM-CO score (Changoor et al.Osteoarthritis Cartilage 2011;19:126–35), and zonal proportions measured. SEM images were captured from locations matched to PLM. Fibre orientations were assessed in SEM and compared to those observed in PLM. CO was also assessed in individual SEM images and combined to generate a SEM-CO score for overall CO analogous to PLM-CO. Fibre diameters were measured in SEM. PLM-CO and SEM-CO scores were correlated, r = 0.786 (P < 0.00001, n = 32), after excluding two outliers. Orientation observed in PLM was validated by SEM since PLM/SEM correspondence occurred in 91.6% of samples. Proportions of the deep (DZ), transitional (TZ) and superficial (SZ) zones averaged 74.0 ± 9.1%, 18.6 ± 7.0%, and 7.3 ± 1.2% in normal, and 45.6 ± 10.7%, 47.2 ± 10.1% and 9.5 ± 3.4% in degraded cartilage, respectively. Fibre diameters in normal cartilage increased with depth from the articular surface [55.8 ± 9.4 nm (SZ), 87.5 ± 1.8 nm (TZ) and 108.2 ± 1.8 nm (DZ)]. Fibre diameters were smaller in repair biopsies [60.4 ± 0.7 nm (SZ), 63.2 ± 0.6 nm (TZ) and 67.2 ± 0.8 nm (DZ)]. Degraded cartilage had wider fibre diameter ranges and bimodal distributions, possibly reflecting new collagen synthesis and remodelling or collagen fibre unravelling. Repair tissues revealed the potential of microfracture-based repair procedures to produce zonal CO resembling native articular cartilage structure. Values are reported as mean ± 95% confidence interval. This detailed assessment of collagen architecture could benefit the development of cartilage repair strategies intended to recreate functional collagen architecture.

Link to Article

http://dx.doi.org/10.1016/j.joca.2011.09.007

Iron deficiency drives an autosomal dominant hypophosphatemic rickets (ADHR) phenotype in fibroblast growth factor-23 (Fgf23) knock-in mice

Authors

Emily G. Farrow, Xijie Yu, Lelia J. Summers, Siobhan I. Davis, James C. Fleet, Matthew R. Allen, Alexander G. Robling, Keith R. Stayrook, Victoria Jideonwo, Martin J. Magers, Holly J. Garringer, Ruben Vidal, Rebecca J. Chan, Charles B. Goodwin, Siu L. Hui, Munro Peacock, and Kenneth E. White

Abstract

Autosomal dominant hypophosphatemic rickets (ADHR) is unique among the disorders involving Fibroblast growth factor 23 (FGF23) because individuals with R176Q/W and R179Q/W mutations in the FGF23 176RXXR179/S180 proteolytic cleavage motif can cycle from unaffected status to delayed onset of disease. This onset may occur in physiological states associated with iron deficiency, including puberty and pregnancy. To test the role of iron status in development of the ADHR phenotype, WT and R176Q-Fgf23 knock-in (ADHR) mice were placed on control or low-iron diets. Both the WT and ADHR mice receiving low-iron diet had significantly elevated bone Fgf23 mRNA. WT mice on a low-iron diet maintained normal serum intact Fgf23 and phosphate metabolism, with elevated serum C-terminal Fgf23 fragments. In contrast, the ADHR mice on the low-iron diet had elevated intact and C-terminal Fgf23 with hypophosphatemic osteomalacia. We used in vitro iron chelation to isolate the effects of iron deficiency on Fgf23 expression. We found that iron chelation in vitro resulted in a significant increase in Fgf23 mRNA that was dependent upon Mapk. Thus, unlike other syndromes of elevated FGF23, our findings support the concept that late-onset ADHR is the product of gene–environment interactions whereby the combined presence of an Fgf23-stabilizing mutation and iron deficiency can lead to ADHR.

Link to Article

http://dx.doi.org/10.1073/pnas.1110905108

A murine model of neurofibromatosis type 1 tibial pseudarthrosis featuring proliferative fibrous tissue and osteoclast-like cells

Authors

Jad El-Hoss MSc1,2,*, Kate Sullivan PhD1,2, Tegan Cheng1, Nicole YC Yu BEng(Hons)1, Justin D Bobyn BSc, MBBS2, Lauren Peacock1, Kathy Mikulec1, Paul Baldock PhD3, Ian E Alexander MBBS, FRACP, PhD2, Aaron Schindeler PhD1,2, David G Little MBBS, FRACS(Orth), PhD

Abstract

Neurofibromatosis type 1 is a common genetic condition caused by mutations in the NF1 gene. Patients often suffer from tissue specific lesions associated with local double-inactivation of NF1. In this study, we generated a novel fracture model to investigate the mechanism underlying congenital pseudarthrosis of the tibia (CPT) associated with NF1. We used a Cre-expressing adenovirus (AdCre) to inactivate Nf1 in vitro in cultured osteoprogenitors and osteoblasts, and in vivo in the fracture callus of Nf1flox/flox and Nf1flox/- mice. The effects of the presence of Nf1null cells were extensively examined. Cultured Nf1null committed osteoprogenitors from neonatal calvaria failed to differentiate and express mature osteoblastic markers, even with rhBMP-2 treatment. Similarly, Nf1null inducible osteoprogenitors obtained from Nf1 mouse muscle were also unresponsive to rhBMP-2. In both closed and open fracture models in Nf1flox/flox and Nf1flox/- mice, local AdCre injection significantly impaired bone healing, with fracture union being <50% that of wild type controls. No significant difference was seen between Nf1flox/flox and Nf1flox/- mice. Histological analyses showed invasion of the Nf1null fractures by fibrous and highly proliferative tissue. Mean amounts of fibrous tissue were increased upwards of 10-fold in Nf1null fractures and BrdU staining in closed fractures showed increased numbers of proliferating cells. In Nf1null fractures, TRAP+ cells were frequently observed within the fibrous tissue, not lining a bone surface. In summary, we report that local Nf1 deletion in a fracture callus is sufficient to impair bony union and recapitulate histological features of clinical CPT. Cell culture findings support the concept that Nf1 double inactivation impairs early osteoblastic differentiation. This model provides valuable insight into the pathobiology of the disease, and will be helpful for trialing therapeutic compounds.

Link to Article

htt://dx.doi.org/10.1002/jbmr.528/