Reconstruction of long-bone segmental defects remains a difficult problem for orthopedic surgeons. While metallic implants and allografts are two clinically available options for reconstruction, they are fraught with complications such as implant loosening, nonunion, and microfracture. Tissue engineering may soon introduce alternative treatment options by offering osteoinductive biomaterial scaffolds in lieu of the metallic implants or biologic grafts. There are at least two strategies by which osteoinductive properties can be conferred onto biomaterial scaffolds. The first strategy involves engraftment of mesenchymal stem cells on the scaffolds to provide a viable bone substitute. While the efficacy of this approach has been demonstrated,14
many concerns regarding the cell sourcing, effective and reproducible engraftment protocols, and cost have hindered its clinical applicability. The second strategy is to introduce osteogenic factors (recombinant or animal-derived)15–18
or genes (plasmids or vectors) onto the scaffold for localized delivery.3,19
The localized delivery approach has shown promising results in preclinical studies and has come to fruition in surgical applications such as spinal fusion scaffolds (cages) augmented by bone morphogenetic proteins.20
However, the complications associated with the need for high local doses of bone morphogenetic proteins21–25
and the safety concerns surrounding viral-based gene delivery26–28
have thus far limited wide clinical applicability of this strategy. In this study, we tested the alternative approach to enhance bone regeneration around a structural biomaterial scaffold implanted in a massive segmental femoral defect via systemic injections of teriparatide, and demonstrated that this treatment, independent of scaffold's mineral content, led to significant enhancements of mineralized tissue volume, mineral content, union incidence, and biomechanical (torsional) strength and rigidity.
Although teriparatide is primarily approved in the United States for the treatment of osteoporosis in postmenopausal women and in men at high risk for fracture,29
there are ample preclinical and clinical evidence to suggest that different forms of PTH can accelerate fracture healing, and could be beneficial in treating nonunions and delayed unions,10
and in enhancing bony ingrowth in orthopedic implants.30
The anabolic effects of intermittent PTH treatment on the repair of long bone fractures in an animal model were first reported in a rat tibial fracture model in 1999.31
Similar results have since been published confirming that intermittent PTH treatment increases fracture callus volume and improves the biomechanical properties of the fractured bone,9,32–38
even after withdrawal of treatment.9,33
The effects of hPTH(1,34) on bone regeneration in critical-sized osteotomy models were first reported by Bonadio et al.
wherein DNA plasmids encoding the active fragment of hPTH(1,34) were loaded onto lyophilized collagen sponges, and then implanted in critical-size (up to 2.0
cm long) defects in the tibiae of beagle dogs. This study reported that high doses (100
mg) of hPTH(1,34) encoding plasmids were necessary for progressive bone filling in the defect, although no incidence of complete gap filling were reported, whereas lower doses (1.0–20
mg) showed no substantial bone regeneration.3
In a follow-up study, Chen et al.
investigated the effects of daily injections (40
μg/kg body weight) of rhPTH(1,34) on bone regeneration in a rat model of critical size (4
mm) femoral defects, with or without localized, collagen matrix-mediated gene delivery of hPTH(1,34).4
At 6 weeks postsurgery, neither treatment on its own resulted in increased BMD and BMC at the osteotomy site. However, compared to local gene delivery of hPTH(1,34), the systemic injections of rhPTH(1,34) resulted in increased defect filling based on planar X-ray quantification. Interestingly, the combined treatment of systemic and localized PTH resulted in significant increases in BMD, BMC, and percent bone regeneration in the defect, compared to either treatment alone, or untreated controls.4
However, as in the previous work, no complete defect filling or union was observed. While these studies bear similarities to our study, there are major differences in the femoral defect model, the scaffold, the timing of treatment, and the functional assessment of the treatment outcome. In our study, we utilized a mouse model of a 4
mm femoral defect, which measures close to 20%–25% of the entire length of the femur, and thus models a massive segmental defect as opposed to critical nonunion defects in larger animals. In addition, our PLA and PLA/β-TCP scaffolds are structurally rigid and load bearing as they were stabilized using intramedullar pins, compared to the nonstructural collagen matrices stabilized with external fixators.3,4
This is a significant advantage of our model since the combination of PTH and in vivo
mechanical stimulation has been shown to synergistically increase bone formation rates.39
Further, the systemic injections of teriparatide in our study were initiated at 1 week postreconstruction, and continued for 8 weeks until sacrifice, to simulate delayed onset of treatment that would commence when nonunion is clinically evident. Finally, and to the best of our knowledge, our study is the first to report functional outcomes that demonstrated increased bone regeneration and incidence of union based on 3D micro-CT data, and enhanced biomechanical properties (torsional strength and rigidity) in massive femoral defects following tissue-engineered limb reconstruction and daily systemic injections of teriparatide.
Regardless of the scaffold used in the femoral reconstruction, only a subset of specimens (30%) treated with teriparatide formed a bridging bone union across the segmental defect, with significant torsional rigidity and strength. None of the saline-injected controls, on the other hand, displayed union or comparable torsional stability. The reasons for this variability in the response to teriparatide treatment are unclear at this time, but might be related to slight variability in animal weights, which may have led to undocumented dosing variability, surgical factors related to the rigidity of the scaffold stabilization using an intramedullary pin or inadvertent injury to the periosteum of the femur during surgical osteotomy and reconstruction, and/or the inherent variability in experimental animals. It is possible that optimizing the teriparatide dose and window of treatment (i.e., timing of onset and withdrawal of treatment) might lead to more consistent and reproducible bridging bone repair in the treated animals, which warrants formal investigation in future experiments.
Interestingly, the observed teriparatide-induced increases in the callus volume of the PLA scaffolds were lost between 6 and 9 weeks, but remained persistent in the PLA/β-TCP scaffolds. This might suggest a synergistic effect of the embedded β-TCP particles with teriparatide. The mechanism of this phenomenon is not clear but may be related to enhanced osteoblast adhesion on the β-TCP particles,40
which might increase their mechanosensitivity to the in vivo
loading environment. This possibility is supported by reports that suggest that the activity of transcriptional attenuators of the anabolic response of osteoblasts to PTH such as the nuclear matrix protein 4/cas interacting zinc finger or Nmp4/CIZ is sensitive to cell adhesion and can be suppressed in response to mechanical perturbations.41
One of the limitations in our study is that we did not probe the mechanism of action of PTH and the reason of the observed synergy with scaffold mineral content using molecular and cellular assays over time. This should be the subject of future studies, especially since the anabolic pathways in bone regeneration at sites of fracture or osteotomy in response to PTH have yet to be fully identified. Endogenous PTH acts as a regulatory hormone for serum calcium homeostasis. The N-terminus of endogenous PTH activates the PTH-1 receptor (a G protein-coupled receptor), and activates the cyclic AMP-dependent protein kinase A and calcium-dependent protein kinase C pathways to regulate osteoblast function.29
Interestingly, continuous treatment with PTH is reportedly associated with a decrease in BMD and hypercalcemia (which mimics chronic elevation of endogenous PTH levels due to hyperparathyroidism). Sclerostin (a negative regulator of osteogenesis transcribed by the Sost
gene) is a Wnt/β-catenin antagonist produced by osteocytes.42,43
Recent findings have shown that continuous elevation of PTH downregulates Sost
, leading to increased osteoblast apoptosis. On the other hand, the anabolic effects of intermittent PTH dosing are associated with attenuation of osteoblast apoptosis independent of Sost.42
regulation plays a role in PTH-stimulated bone repair has yet to be investigated. However, the likely targets of the early effects of PTH during repair might include mesenchymal stem cells, periosteal cells, and osteoblast progenitors. Chen et al.
reported positive mRNA expression of PTH-1 receptor at the site of femoral osteotomy, regardless of local or systemic PTH treatment. However, systemic PTH treatment led to increased osteocalcin (Ocn
) mRNA expression in sera and callus tissue at the site of femoral osteotomy.4
Kaback et al.
reported that systemic teriparatide upregulates the transcription factor Osterix (Osx
) in femoral fracture callus.8
Others have shown that PTH effects on fracture repair are associated with enhanced chondrogenesis (chondrocyte recruitment and maturation) in the early fracture callus, mediated in part by canonical Wnt signaling.44
Interestingly, while intermittent PTH treatment stimulates bone growth in vivo
, possibly by increasing local bone factors, its effects on angiogenesis are much less studied despite the critical role of angiogenesis in the process of bone repair. It has been shown that continuous treatment with PTHrP(1–34) increases vascular endothelial growth factor mRNA in human osteoblastic cells from trabecular bone,45
whereas other reports have shown that vascular endothelial growth factor-A production during osteoblast differentiation is inhibited by PTH-related peptide that also inhibited preosteoblast differentiation.46
Others have reported anti-angiogenic effects of PTH in the skin.47
These reports suggest an intriguing mechanism of action for systemic PTH in bone regeneration that might involve negative regulation of angiogenesis, which merits further investigation in future studies.
In conclusion, the current findings suggest that the effects of intermittent, systemic PTH in scaffold mediated bone repair and regeneration could be enhanced by mineralized scaffold components such as β-TCP. Further studies delineating the mechanism of the observed synergy between PTH and β-TCP are warranted using in vitro and in vivo model systems that can potentially lead to tissue-engineered alternatives to bone graft reconstruction.