Session II - Basic Science

Prolonged Expression of Insulin-Like Growth Factor-I and Platelet-Derived Growth Factor-A in the Fracture Callus of Adult Rats

Ralph A. Meyer, Jr., PhD; Bhaloo J. Desai, PhD; Martha H. Meyer, MS; Scott Porter, MD; James F. Kellam, MD, Orthopaedic Research Laboratory, Carolinas Medical Center, Charlotte, NC (supported by OTA Research Grant)

Purpose: The rate of fracture healing slows with age in both humans and rats. The reason for this is not clear. Recent data implicate embryonic growth factors that stimulate bone induction in the stimulation of fracture repair. However, fracture healing in adult rats, although slower than in young rats, is not accompanied by prolonged expression of either Indian hedgehog or any of the bone morphogenetic proteins. This led to a broader search for other skeletally related cytokines that may be involved in the formation of bridging callus. We tested for quantitative as well as temporal changes in mRNA gene expression.

Methods: Intact female Sprague-Dawley rats at 6 and 26 weeks of age were anesthetized. An intramedullary rod was placed in the left femur, and a closed mid-diaphyseal fracture was induced. Intact rats of the same age were used for 0 time controls. Rats were euthanized at 1, 2, 4, 6, 8, and 10 weeks after fracture (n = 6/age/time point). Radiographs were made at euthanasia. The femora were rapidly harvested, and the fracture calluses were frozen in liquid nitrogen and stored at ­80 C. The calluses were homogenized with a Brinkman Polytron, and total RNA was extracted with TRIzol. Aliquots (0.8 mg) of the RNA were reverse transcribed (RT) using random hexamer primers. The cDNA was amplified by polymerase chain reaction (PCR) using primers specific for the rat genes of interest. The amplimers were separated by electrophoresis and blotted to nylon membranes (Southern blots). The blots were probed with ³²P-labeled internal oligonucleotides specific for each amplimer. The radioactivity was quantified with a phosphor imager, and the data were summarized as mean ± SEM. Comparisons were made with the Mann-Whitney U test or the t test.

Results: Most (73%) fractures were simple transverse midshaft femoral fractures (32-A3.2). A few (12%) were oblique (32-A2.2), and others (15%) had a wedge-shaped fragment (32-B2.2). The latter two categories were assigned uniformly to the treatment groups and did not seem to affect either healing or gene expression. All young rats achieved bridging callus with remodeling by the fourth week, whereas the adult rats required 8 to10 weeks. All genes studied increased in mRNA expression after fracture, reaching a peak at 1 to 2 weeks after fracture. All genes in the young rats and most genes in the adult rats subsided to baseline by 4 weeks after fracture. Only bone matrix genes (osteocalcin, type I collagen, and bone sialoprotein) and genes related to angiogenesis (VEGF [VEGF₁₂₀, VEGF₁₆₄, and VEGF₁₈₈], Flk-1, and angiopoietin-1) had significantly prolonged expression at 4 and 6 weeks after fracture in the adult rats. Indian hedgehog, TGF-b1, and BMP-2, -4, -6, and -7 had the same expression pattern in young and adult rats with a peak at 1 to 2 weeks after fracture and subsidence to baseline by 4 weeks after fracture. In contrast, insulin-like growth factor-I (P < 0.05) and perhaps platelet-derived growth factor-A (P = 0.1) had greater mRNA expression at 4 weeks after fracture in adult animals than in younger animals.

Discussion: What causes the progressive lag in the healing rate with age is not clear. This lag provides an opportunity to elucidate the genes required for the periosteal reaction and for bridging-callus formation. Such genes should show a prolonged expression pattern in adult rats to accompany the prolonged matrix synthesis. The femoral fractures in adult rats do heal, both radiographically and biomechanically, albeit more slowly than in 6-week-old rats. To accomplish this slower healing, there was a prolonged expression of osteocalcin, bone sialoprotein, and type-I collagen in the adult samples, which reflected the continued bone matrix synthesis as the healing progressed. Because osteogenesis requires vascular proliferation, there was also continued expression of genes related to angiogenesis in the adult animals. Despite this extended bone synthesis in the adult animals, many of the skeletally active cytokines (Indian hedgehog, BMP-2, BMP-4, BMP-6 and BMP-7) were not significantly prolonged in their elevated mRNA expression. These genes increased to a peak expression at 1 week after fracture and subsided thereafter. None was present at 4 or 6 weeks after fracture to accompany the prolonged matrix synthesis, which occurred in the adult rats. The lack of change in gene expression between the two ages studied suggests that neither the rate of the periosteal reaction nor the rate of bone formation to bridge the fracture gap seems to influence the time of expression of this group of cytokines. It may be that the injury stimulates an elevation in the expression of these genes that will peak and subside within about two weeks regardless of the state of healing of the fracture. It may also be that this early stimulus is needed for formation of the soft callus immediately after injury and not for the later stages of bone synthesis. A different set of cytokines would seem to be needed to stimulate the later stages of bony healing. Two cytokine genes, insulin-like growth factor-I and platelet-derived growth factor-A, were expressed for longer periods of time in the adult rats. This suggests a role for these two genes in the formation of bridging callus. Further study will undoubtedly reveal other genes that stimulate the later stages of fracture healing.

Conclusions: Mid-diaphyseal fracture of the femur of a young or adult rat is followed by increased mRNA gene expression for many cytokine genes. Most follow the same gene expression pattern at both ages. In adults, prolonged gene expression is needed to stimulate the slower formation of bridging callus. Insulin-like growth factor-I and platelet-derived growth factor-A may play a role in the stimulation of the later stages of fracture healing.