Are endogenous BMPs necessary for bone healing during distraction osteogenesis? - PubMed (original) (raw)

Are endogenous BMPs necessary for bone healing during distraction osteogenesis?

Norine Alam et al. Clin Orthop Relat Res. 2009 Dec.

Abstract

Previous reports suggest the application of exogenous BMPs can accelerate bone formation during distraction osteogenesis (DO). However, there are drawbacks associated with the use of exogenous BMPs. A possible alternative to the use of exogenous BMPs is to upregulate the expression of endogenous BMPs. Since DO results in spontaneously generated de novo bone formation in a uniform radiographic, histological, and biomechanical temporal sequence, a genetically engineered model lacking endogenous BMP2 should have measurable deficits in bone formation at different time points. We performed DO on BMP2(fl/+) and BMP2(fl/+ cre) mice using a miniature Ilizarov fixator. Distracted samples were collected at various time points and analyzed using real time-quantitative PCR, lCT, radiology, immunohistochemistry, histology, and biomechanical testing. Immunohistochemical studies of 34-day heterozygous samples showed reduced expression of BMP2, BMP7, BMPR1a, ACTR1, and ACTR2b. lCT analysis of 51-day heterozygous samples revealed a decrease in trabecular number and increase in trabecular separation. Biomechanical testing of 51-day heterozygous samples revealed decreased stiffness and increased ultimate displacement. Radiological analysis showed the heterozygotes contained a decreased bone fill score at 17, 34, and 51 days. These data suggest endogenous BMPs are important for bone healing and manipulating endogenous BMPs may help accelerate bone consolidation during DO.

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Figures

Fig. 1

Fig. 1

Experimental design of the 54 operated mice. Distracted tibial samples were collected from wild-type and heterozygous mice at various time points: 11, 17, 34, and 51 days post-surgery. These samples were analyzed using RT-qPCR, μCT, Faxitron xray, immunohistochemistry, Goldner-Trichrome staining (histology), and biomechanical testing.

Fig. 2

Fig. 2

Nonoperated mice contain reduced BMP2 expression in the limbs. mRNA was collected from the limbs of non-operated 1-week-old wild-type, heterozygous and homozygous mice and analyzed using RT-qPCR. GAPDH was used as an endogenous control. Decreased BMP2 levels were expressed in the heterozygous and homozygous mice compared to the wild-type mice. No change in BMP2 levels was detected between the wild-type and heterozygous mice.

Fig. 3A–P

Fig. 3A–P

μCT and radiological images of distracted limbs collected from wild-type and heterozygous BMP2 knockout mice. Distracted tibial samples were collected at 11 days (mid-distraction phase), 17 days (end of distraction phase), 34 days (early consolidation phase), and 51 days (end of consolidation phase) post-surgery and analyzed using (A–H) μCT and (I–P) Faxitron xray.

Fig. 4A–B

Fig. 4A–B

Static histomorphometric parameters revealed heterozygous mice contained less bone formation at 51 days. Distracted samples collected at 51 days post-surgery revealed an increase (p = 0.0093) in (A) trabecular separation and decrease (p = 0.0091) in (B) trabecular number, indicating there was less bone formation in the heterozygous mice during the end of consolidation compared to the controls.

Fig. 5A–H

Fig. 5A–H

BMP2 expression in chondrocytes present in the distracted limbs of BMP2 knockout mice. Immunohistochemical images of (A, B, E, F) 34 and (C, D, G, H) 51 day heterozygous and wild-type distracted samples revealed that the heterozygotes contained decreased BMP2 expression at 34 days (early consolidation) that gradually leveled off by 51 days (late consolidation). BMP2-stained chondrocytes are indicated by the arrows in the diagram. (Immunohistochemistry, BMP2; original magnification, ×10 and ×40).

Fig. 6A–D

Fig. 6A–D

Histological images of distracted limbs of wild-type and heterozygous mice at 51 days post-surgery are shown. MMA-embedded tibial sections of (A, C) wild-type and (B, D) heterozygous mice were stained using the Goldner-Trichrome technique. At 51 days (end of consolidation phase), both groups contained varying levels of mineralized (green) and nonmineralized (red) tissue. Chondrocytes and fibrous tissue were also present in the distracted samples, as indicated by the arrows in the diagram. (Stain, Goldner-Trichrome; original magnification, ×25 and ×100).

Fig. 7A–B

Fig. 7A–B

Biomechanical testing results of distracted wild-type and heterozygous BMP2 knockout mice at 51 days post-surgery are shown. The three-point bending test was performed on distracted wild-type and heterozygous samples collected at 51 days (end of consolidation phase) post-surgery. A decrease (p = 0.323) in (A) stiffness and increase (p = 0.654) in (B) ultimate displacement was observed in the heterozygous mice compared to the wild-type controls.

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