Aim 2: Assess the numbers of progenitor cell colony-forming units (CFU) and osteogenic cells per CFU in bone marrow and periosteum tissues from osteotomized rats in simulated weightlessness versus normal gravity.

Aim 3: Determine the rate and extent of fracture healing in osteotomized rats in simulated weightlessness when treated with PTH or bisphosphonate therapy.

Aim 4: Assess the numbers of progenitor cell CFU and osteogenic cells per CFU in bone marrow and periosteal tissues from osteotomized rats in simulated weightlessness undergoing either PTH or bisphosphonate therapy.

In year 1, rats experiencing normal gravity (weight bearing or WB) exhibited a spontaneous repair of cortical bone trauma within a 5-week healing period. Intermittent PTH therapy enhanced this WB repair process by: 1) increasing the rate of hard callus formation and peak hard callus volume compared to vehicle-control rats; 2) increasing bone formation rates (BFR); and 3) increasing the number of osteoprogenitors within bone marrow tissue.

In year 2, rats experiencing hind limb unloading (non-weight bearing or NWB) exhibited a greatly diminished level of spontaneous repair of bone trauma as compared to the WB group within the same healing period. This was reflected in a diminished maximum hard callus volume and a rate of hard callus formation that were both only ~20% of those of the WB group. Reacquisition of a weight-bearing status equal in time to that of the period of simulated weightlessness prior to bone trauma (NWB-WB group) did not substantially improve the bone healing response. In this case, this group exhibited a maximum hard callus volume that was only 34% of, and a rate of hard callus formation that was only 28% of the WB group. Both NWB and NWB-WB groups exhibited low bending strength values at their respective callus sites by 5-weeks post-op (0.28 ± 0.05 N/m) in stark contrast to the bending strength of the WB callus (1.75 ± 0.35 N/m). Histology revealed that 46% of the NWB group fibulae were reconnected by a fibrous soft tissue union (“non-union”), while all of the WB group specimens were reconnected by a bony tissue union. Lastly, NWB reduced the number of marrow progenitor cells and osteoprogenitors by 90% from the levels exhibited by WB bone specimens.

In year 3, intermittent PTH therapy (80 ug/kg BW) was shown to partially reverse the deleterious effects of NWB on cortical bone trauma healing. Only 12.5% of NWB-PTH specimens were classified as “non-unions” (a 73% decrease from that of NWB) and 50-75% increases in maximum hard callus volume and hard callus formation rates were observed as compared to NWB specimens. PTH therapy increased BFR by ~2-fold over those exhibited by NWB rats. PTH therapy enhanced the numbers of osteoprogenitor cells by ~4-fold as compared to those measured from NWB specimens. We are currently assessing bending strength and histology of these specimens. Altogether, intermittent PTH therapy partially restores select aspects of bone healing under simulated microgravity. Grant BL00405 was extended for an extra 6 months of work (04/01/07 – 09/01/07) and during this time NWB rats were treated with alendronate (bisphosphonate). Currently, the results from this trial are under analysis and results will be forth coming.

Findings from BL00405 indicate that cortical bone fracture healing is impaired in hind limb unloaded rats. The implication of these findings is that bone trauma repair in astronauts on long space missions would likely be compromised, and presents a potential threat to mission effectiveness and astronaut health. Further, these findings also suggest that astronauts who return from extended spaceflight missions are at continued risk for impaired bone trauma repair after returning to a normal gravity environment even up to a period of time equal to that of the space mission.

Secondly, findings from BL00405 indicate that extended exposure to hind limb unloading results in drastic reductions in both total marrow progenitor cells and osteoprogenitors. Thus, a simulated microgravity situation appears to cause a reduction in the number of preosteoblasts that would be needed to generate a functional osteoblast population needed to repair bone trauma. These findings provide a plausible mechanism of action underlying this impaired fracture healing response. Thirdly, findings from BL00405 indicate that intermittent PTH therapy improves fracture healing under chronic hind limb unloading, though it does not normalize fracture healing to WB levels. The dramatic reduction in the number of fracture “non-unions” resulting from PTH therapy suggests that this treatment is a viable candidate to be tested as a countermeasure to offset any potential deleterious effects of weightlessness on bone trauma healing. Fourthly, findings from BL00405 indicate that PTH therapy significantly increased the number of functional osteogenic progenitor cells in bone under chronic hind limb unloading. PTH therapy’s increase in the number of osteoprogenitors correlated with its improvements in fracture healing in NWB rats strongly suggests a cause and effect between these parameters and strengthens our hypothesis that a decrease in progenitor cells represents a mechanism of action underlying the impairment to fracture healing. If correct, then the implications of these findings are that marrow progenitor cell populations may be altered in astronauts on long space missions and might manifest in deficiencies in musculoskeletal tissue repair after trauma. Given that BL00405 has reached is closure date, our remaining plans are to complete the remaining data assessments and submit manuscripts for submission to peer-reviewed publications.

Our studies found that PTH therapy significantly increased the prevalence of osteogenic progenitor cells within bone marrow tissue over that measured in vehicle-control rats (normal gravity group). Further, PTH therapy increased the prevalence of osteogenic progenitor cells within bone marrow tissue 4-fold over that measured in vehicle-control hind limb suspended rats (chronic non-weight bearing). Coupled with the observations that PTH therapy improved fracture healing in hind limb suspended rats, these findings suggest that an underlying mechanism of action of PTH in its anabolic activity for enhancing bone fracture repair lies at the level of increasing the pool size of functional bone cells derived from osteogenic precursor cells. These same findings suggest that PTH therapy should aid in the healing of recalcitrant bone fractures here on Earth particularly in patients experiencing paralysis, chronic immobility or extended bed rest.

(1) task progress shows that prolonged exposure to hind limb unloading resulted in impaired fibular fracture healing. This directly addresses CPR #2.11: Does prolonged exposure to hypogravity lead to impaired healing of fractures? Our work raises the CRL of this enabling question from an original 1-2 to a 3-4 level.

(2) task progress shows that prolonged exposure to hind limb unloading decreased the population size of bone marrow osteoprogenitor cells. This directly addresses CPR #2.23: Does hypogravity affect the size, viability, or differentiation of precursor bone cell populations? Our work raises the CRL of this enabling question from an original 1 to a 2-3 level.

(3) task progress shows that prolonged exposure to hind limb unloading decreased the osteogenic differentiation of bone marrow osteoprogenitor cells. This directly addresses CPR #2.23: Does hypogravity affect the size, viability, or differentiation of precursor bone cell populations? Our work raises the CRL of this enabling question from an original 1 to a 2-3 level.

(4) task progress shows that PTH therapy improves bone healing outcomes incurred by prolonged exposure to hind limb unloading (CPR #2.06: What pharmacological agents will most effectively minimize the decrease in bone mass with hypogravity? Are anabolic as well as anti-resorptive agents required?). Our work raises the CRL of this enabling question from an original 1 to a 2-3 level.

(5) task progress shows that PTH therapy enhances bone formation rates in rats exposed to chronic hind limb unloading thereby boosting the rate of bone production under simulated weightlessness (CPR #2.06: What pharmacological agents will most effectively minimize the decrease in bone mass with hypogravity? Are anabolic as well as anti-resorptive agents required?). Our work raises the CRL of this enabling question from an original 1 to a 2-3 level. (6) task progress shows that PTH therapy improves the population size of marrow osteoprogenitors in rats exposed to chronic hind limb unloading (CPR #2.06: What pharmacological agents will most effectively minimize the decrease in bone mass with hypogravity? Are anabolic as well as anti-resorptive agents required?). Our work raises the CRL of this enabling question from an original 1 to a 2-3 level.

Altogether, our findings suggest that fracture healing may be adversely affected by chronic exposure to weightlessness, and PTH injections, as an anabolic bone therapeutic approach, may be used to ameliorate some of these adverse effects on bone trauma healing.

Summaries and Abstracts, 36th International Sun Valley Workshop on Skeletal Tissue Biology, Sun Valley, ID, July 30 - Aug 3, 2006. p. 18-20. , Aug-2006

Aim 1: Assess the rate and extent of fracture healing in osteotomized rats in simulated weightlessness versus normal gravity. Aim 2: Assess the numbers of progenitor cell colony-forming units (CFU) and osteogenic cells per CFU in bone marrow and periosteum tissues from osteotomized rats in simulated weightlessness versus normal gravity. Aim 3: Determine the rate and extent of fracture healing in osteotomized rats in simulated weightlessness when treated with PTH or bisphosphonate therapy. Aim 4: Assess the numbers of progenitor cell CFU and osteogenic cells per CFU in bone marrow and periosteal tissues from osteotomized rats in simulated weightlessness undergoing either PTH or bisphosphonate therapy.

2) Key Findings

In year 1, as assessed by in vivo micro-computed tomography (micro-CT), a normal gravity group (weight bearing or WB) exhibited spontaneous repair of bone trauma within a 5-week healing period. PTH therapy enhanced this repair rate by increasing: 1) the rate of hard callus formation and peak volume of hard callus by ~50% over that of vehicle-control rats; and 2) the volume of cortical bone adjacent to the trauma site by ~60%. PTH therapy also increased cancellous bone content within the healing callus by ~40% over that of control rats. Mineral apposition rates were increased in PTH-treated rats as compared to control rats. PTH therapy increased the prevalence of osteogenic cell CFUs within bone marrow tissue by ~80% over that measured in vehicle-control WB rats. These findings provide support for the validity of Hypothesis 4 (PTH therapy will significantly increase the number of functional osteogenic progenitor cells in bone under simulated weightlessness).

In this second year, we quantified the extent of bone trauma repair associated with simulated weightlessness (non-weight bearing or NWB). As assessed by in vivo micro-CT, the NWB group exhibited a greatly diminished level of spontaneous repair of bone trauma as compared to the WB group within the same healing period. This diminished level was reflected in a maximum hard callus volume that was only 22% of, and a rate of hard callus formation that was only 19% of the WB group. Reacquisition of a weight-bearing status equal in time to that of the period of simulated weightlessness prior to bone trauma (NWB-WB group) did not substantially improve the bone healing response. In this case, this group exhibited a maximum hard callus volume that was only 34% of, and a rate of hard callus formation that was only 28% of the WB group.

Neither the NWB nor the NWB-WB groups exhibited measurable bending strength at the callus site by 5-weeks post-op. This finding is in stark contrast to the bending strength of the WB callus, which was measured to be 4.74 ± 1.95MPa (n=10 bones) or roughly half the bending strength of undamaged fibulae (9.57 ± 2.05MPa). Histological analysis revealed that the NWB group fibulae were reconnected by a fibrous soft tissue union, while the NWB-WB group was reconnected by a cartilaginous soft tissue union.

Our ongoing studies indicate that intermittent PTH at doses higher than those used to prevent osteoporosis can reverse the deleterious effects of simulated weightlessness on bone trauma healing. Our most recent experiments found that 120-mg/kg body weight dosage given 5 times per week to NWB rats yielded a bony union across the original osteotomy site that was roughly 50% the cancellous bone volume of the WB group over the same healing period. We are currently assessing bending strength and histology of these specimens. Altogether, these findings suggest that intermittent PTH therapy may be a candidate countermeasure against this risk of impaired bone healing in space.

3) Impact of Findings

The findings obtained within the first two years of funding support the validity of Hypothesis 1 (Native fracture healing will be adversely affected by simulated weightlessness). The implication of these findings is that bone trauma repair in astronauts on deep-space missions would likely be compromised, and presents a potential threat to mission effectiveness and astronaut health. Further, these findings also suggest that astronauts who return from extended spaceflight missions are at continued risk for impaired bone trauma repair after returning to a normal gravity environment even up to a period of time equal to that of the space mission.

Our preliminary findings suggest that the number of progenitor cell CFU is not expected to be reduced in NWB rats. Rather, the number of CFU expressing an osteogenic phenotype in bone marrow in vivo is likely reduced in NWB rats compared to WB rats. Thus, a simulated weightlessness situation may be caused by a reduction in the existing number of functional preosteoblasts and osteoblasts available for immediate use to repair bone trauma. This finding would support the validity of Hypothesis 2 (The population of functional osteogenic progenitor cells in bone will be significantly reduced by simulated weightlessness). Lastly, our results indicate that our Hypothesis 3 (PTH therapy will significantly improve fracture healing under simulated weightlessness) has merit. The implication of these findings is that PTH therapy represents a good candidate to be tested as a countermeasure to offset the deleterious effects of weightlessness on bone trauma healing.

4) Year 3 Proposed Research Plan:

Our immediate plans are to complete the currently acquired data assessments to answer definitively the goals of all of our specific aims. This includes the completion of a triplicate trial in order to meet our statistical power requirements. Further, in the upcoming year we will fully explore a potential countermeasure to offset the deleterious effects of weightlessness on bone trauma healing (PTH therapy will significantly improve fracture healing under simulated weightlessness). We are also currently drafting two manuscripts for submission to peer-reviewed publications.

Additionally, our current findings that demonstrate impaired bone healing during and after an extended exposure to simulated weightlessness should manifest relevance to the underlying causes of impaired bone healing in patients experiencing paralysis, immobility or extended bed rest. Our data indicating that intermittent PTH therapy can counteract the impairment of bone healing under a non-weight bearing situation may offer a potential treatment for augmenting bone healing in these Earth-bound, non-weight bearing patients.

A surprising finding in our investigations was the observation that a bone-healing impairment continues well after a simulated weightlessness condition is terminated and a normal weight bearing routine is re-established. Rats that were NWB for 4-weeks were returned to a WB condition for an additional 4-weeks before bone trauma was initiated. In this NWB-WB group of rats the bone trauma healing capacity was only marginally better than that exhibited by the NWB rats, and certainly not of sufficient healing quality to promote proper mechanical function (i.e., bending strength). Overall, when these findings are extrapolated to the human setting, our results would suggest that long bone trauma to astronauts that have returned back to Earth after completing an extended deep space mission will not heal properly. These findings have an additional bearing towards answering CPR question # 2.11a: Does prolonged exposure to hypogravity lead to impaired healing of fractures?

Lastly, our year 1 results and current studies using PTH in the NWB group, indicate that our third Hypothesis (PTH therapy will significantly improve fracture healing under simulated weightlessness) has merit. The implication of these findings is that PTH therapy represents a good candidate to be tested as a countermeasure to offset the deleterious effects of weightlessness on bone trauma healing. These findings have a bearing on answering CPR question #2.06: What pharmacological agents will most effectively minimize the decrease in bone mass with hypogravity? Are anabolic as well as anti-resorptive agents required?

2006 March. , Mar-2006

2005 November , Nov-2005