Large structural allografts are the biomaterial of choice for reconstructing massive long bone defects that might otherwise require prosthetics or amputation. Despite their abundant availability through tissue banks, processed allografts, which are typically devitalized, experience major complications that affect their functional longevity, including non-unions (27–34%) and graft fractures (24–27%) [5
]. The ability of the devitalized allograft to repair micro-damage and establish union with the host is limited due to the lack of osteogenic, angiogenic, and remodeling capabilities [15
]. It is therefore important to design treatment strategies that enhance the functional longevity of processed allografts by addressing the aforementioned biological limitations. Strategies that include cell grafting and localized growth factor or gene delivery have been investigated in preclinical models of massive defects with encouraging results [15
], but to date have not been widely implemented clinically. On the other hand, strategies that involve endocrine hormone treatment (e.g.
parathyroid hormone or PTH), which have shown efficacy in preclinical animal models of fracture repair and are arguably easier to implement clinically, have not been investigated in the context of enhancing structural allograft osseointegration and healing. To address this, the current study investigated the efficacy of teriparatide on functional integration of devitalized allografts in a mouse model of femoral reconstruction.
Our findings demonstrated that daily systemic injection of teriparatide (40 µg/kg body weight) induced persistent cartilage and enhanced trabecular bone formation at 4 weeks (), which led to remarkable graft-host integration at 6-weeks (). Moreover, we observed a significant 2-fold increase in normalized callus volume (1.04 ± 0.3 vs. 0.54 ± 0.14 mm3/mm, p<0.005), and Union Ratio (0.28 ± 0.07 vs. 0.13 ± 0.09; p<0.005), compared to saline treated controls at 6-weeks. Most importantly, the Union Ratio correlated significantly with the yield torque and torsional rigidity (R2=0.59 and R2=0.77, p<0.001, respectively).
The effects of PTH therapy on bone turnover and osteoporotic fractures have been investigated since 1980 [26
]. Numerous preclinical studies have since shown that multiple forms of PTH are capable of accelerating the rate of fracture healing [8
]. Most of these studies used closed (Einhorn) fracture models whose healing is not typically complicated by nonunion. The combination of systematic parathyroid hormone and a local parathyroid hormone gene therapy in non-union osteotomy models has also shown promising efficacy in inducing bone regeneration across a critical size defect [8
]. While only approved as a prophylactic treatment in osteoporotic patients with high fracture risk, off-label clinical use of teriparatide in patients with delayed nonunion fractures has been recently reported with encouraging preliminary outcomes, which led to clinical trials on the use of teriparatide to accelerate fracture healing [13
Teriparatide effects on bone grafts have only recently been reported. Intermittent teriparatide treatment has been shown to increase the strength and bone mineral content in a rat model of freshly harvested, vascularized long bone allograft, either alone or when followed by treatment with the bisphosphonate zoledronic acid [36
]. It has also been reported that intermittent administration of teriparatide accelerates rat spinal arthrodesis after autologous bone grafting [37
]. To the best of our knowledge, our work is the first to investigate the effects of intermittent PTH or teriparatide treatment on critical-size cortical defects reconstructed with devitalized allografts.
Our results are remarkably similar to the PTH effects reported in rodent models of fracture repair and autografts, despite the biological and biomechanical differences in devitalized cortical allograft healing which we previously reported [14
]. However, there is a significant discrepancy between the doses reported in the literature in preclinical animal models (including ours) and the clinical doses in indicated and off-label uses. For example, animal models typically report the use of doses as high as 2.5 to 200 micrograms/kg/day which are 10- to 1000-fold greater than the recommended clinical dose in humans (20 micrograms per day total, which in a 50 to 80kg person translates to 0.25 to 0.4 micrograms/kg/day). Interestingly, studies that addressed dose-dependent effects of PTH in rodents failed to show an effect at the lower doses [10
], which has been attributed to differences in the hormone metabolism between species [11
]. An alternative explanation is that the fracture models used in these studies are not critical defects or nonunion arthrodesis, and typically heal on their own, which confounds any effects a low dose of the drug might exert. Therefore, the appropriate clinically relevant doses of the drug in a challenging bone healing context, such as devitalized allografts, warrants further investigation.
In addition, the timing of the commencement and withdrawal of the treatment are also important. In the scenario of nonunion arthrodesis, the treatment is likely to be delayed following the radiographic diagnosis of nonunion. However, in a scheduled allograft reconstruction, the treatment can be initiated immediately at the time of surgery. Whether immediate treatment is superior to delayed treatment has not been determined and warrants formal investigation. The therapeutic PTH treatment window is important, as it will likely dictate the cell populations that respond to the drug during the multiple stages of the bone healing process. In osteoporosis, PTH has a direct effect on osteoblasts and activates their proliferation and osteocalcin production while suppressing apoptosis [38
], without affecting osteoclast activity [38
]. On the other hand, PTH has been reported to activate the differentiation of bone marrow stromal cells (BMSCs) [40
], and bone lining cells in vivo, without affecting proliferation [41
]. In addition to these effects on intact bone and osteoprecursor cells, PTH has been shown to enhance fracture healing by increasing proliferation of chondroprogenitors which leads to a bigger cartilage template formation [10
], ultimately leading to an increase in callus volume and bone mineral content via accelerated chondrocyte hypertrophy [42
]. Consistent with these observations, daily teriparatide injections, which were initiated one-week post surgery in our model of allograft reconstruction, resulted in persistent cartilage formation at 4 weeks, which led to improved graft-host integration at 6-weeks via a remarkable periosteal and endosteal trabeculated bone callus. Our study, however, has limitations in that it only evaluated a single regimen of teriparatide at a single time point in young animals. Future studies are warranted to investigate teriparatide dose and treatment timing and duration effects on allograft healing at longer time points, and following various intervals of treatment withdrawal in both young and aging animal models.
One of the most important findings of the current work was demonstrating that the Union Ratio, a micro-CT derived biometric of osseointegration between the allograft and host bone [17
], was a significant predictive variable of the effects of teriparatide on the subfailure biomechanical properties of the allografts. More interestingly, we observed that a minimum Union Ratio threshold must be achieved to radiographically determine functional healing as defined by achieving normal torsional rigidity and yield strength with 99% confidence. Whether this Union Ratio threshold value of 0.2 would scale up in larger animals and human patients would have to be verified in future studies. However, despite the strong univariate correlations, there were several outliers that either achieved functional healing (as defined by torsional properties) without achieving the Union Ratio threshold, or that failed to achieve functional healing despite achieving Union Ratio values ≥0.2. These outliers underscore one of the major limitations of the Union Ratio as a measure of functional healing. Specifically, the Union Ratio is a biometric measuring the ratio of connected graft area to the host based on a binary determination of the voxels as either bone or not bone that does not account for any gradation in the mineral density of the union tissue. Future efforts will be dedicated to developing a mineral density-weighted Union Ratio to account for the mineralized state of the union area, which we hypothesize will improve correlations with the biomechanical properties, and eliminate the outliers in radiographically predicting functional healing.
In conclusion, our data illustrate the remarkable potential of teriparatide (PTH) therapy in enhancing allograft healing and integration with the host in a mouse model of massive femoral defect reconstruction. These results should motivate further investigation in a larger animal model to justify future clinical trials for this indication.
- Teriparatide protracts persistent cartilage formation leading to trabeculated bone callus
- Teriparatide enhances osseointegration and biomechanics of devitalized femoral allografts
- Union Ratio measure of osseointegration correlates significantly with torsional biomechanics
- A functional healing criterion could be defined using the non-invasive Union Ratio biometric