NOTE: End date changed to 12/31/2012 per NSBRI (Ed., 5/1/2012)

Note change in Element, Risk, Gap to align with IRP Rev C, per JSC HRP (Ed., 5/25/2011)

In its first year, NSBRI MA01604 has determined by micro-CT imaging and histological analyses that closed femoral fractures in HLU rats exhibit a smaller hard callus healing response compared to weight bearing (WB) counterparts. This suggests that healing of closed femoral fractures in HLU rats is altered. Previously in our project NSBRI BL00405, we determined that an extended period of HLU impaired open fibular fracture healing in rats resulting in a non-union rate of >50% after 6-weeks of healing. However the extent of hard callus healing in HLU closed femoral fractures is of greater magnitude as compared to that exhibited by HLU open fibular fractures.

In its second year, NSBRI MA01604 has confirmed the initial findings that closed femoral fractures in HLU rats exhibit substantially smaller hard callus volumes (40-60% smaller than WB ones) even after 10-weeks of healing. Yet, torsion testing assessments of HLU vs. WB hard calluses indicated sound mechanical properties for both HLU and WB calluses, though the HLU calluses were more brittle. Histological assessments at 10-weeks indicate that the content within the HLU calluses is ~40% mineralizing tissue and ~20% soft/fibrous tissue. Assessments of gene expression and tissue alterations for early fracture healing timepoints (1 and 2 weeks post-fracture) correlating to the chrondrogenic phase (soft tissue callus formation) and the beginnings of the endochondral ossification phases (hard tissue callus formation) of fracture healing are complete. This analysis highlights a delay in endochondral ossification due to lagging chondrocyte hypertrophy, in effect delaying subsequent steps of the healing process such as angiogenic vessel infiltration and mineral deposition. Safranin O histological staining results of proteoglycan deposition within the fracture callus from WB and HLU rats suggested similar amounts of hyaline cartilage tissue in each test group. These histological findings are in agreement with those of gene expression findings whereby aggrecan mRNA levels were similar between groups at 1- and 2-weeks post-fracture. This prolonged chondrocyte maturation step exemplified by delayed hypertrophy, reduced osteo-inductive factor expression, and reduced pro-angiogenic factor expression likely leads to a postponement of the requisite vascularization of HLU callus tissue and its subsequent mineralization. While after a full 10 weeks of healing, it is apparent that HLU fractures heal, the data also indicate that the healing process itself may be altered as compared to fractures from WB rats. In fact, fractures from HLU rats are mechanically sound compared to fractures from WB rats. Yet the explanation for this adaptive healing response in HLU callus is not identified currently.

In its third and fourth years, NSBRI MA01604 utilized pharmaceuticals and biophysical stimulation in attempts to augment fracture healing in rat femora. Both PTH and LIPUS therapies have been shown to enhance the endochondral healing response of femoral fractures under normal gravity conditions. Scl-Ab therapy has also been shown to demonstrate a potent bone-building capacity in rats exhibiting estrogen-depletion osteoporosis and to enhance bone healing responses. Our observations indicate that closed femoral fracture healing response of the treated HLU rats parallels that of the ground based studies and all treatments appear to augment the fracture healing response by the fifth week of healing. Mechanical strength of the hard callus via torsion testing and histo-morphometry of the tissue composition at the fracture site assessments continue to be quantified and characterized. However our overall observations from the raw data indicate that the fracture healing process in treated rats as compared to untreated HLU rats has resulted in calluses with an increased mineralizing tissue content and increased mechanical properties.

Cleveland Clinic Aim 2: The results from this aim were completed and compiled for publication with our collaborators at NASA Ames Research Center (Drs. Ruth Globus and Esther Hill). The manuscript was submitted for review to Acta Astronautica in November 2011 and accepted in April 2012. It is currently in press but available on-line.

Cleveland Clinic and IUPUI Aim 3: All animal trials utilizing the pharmacologic countermeasures (injections of PTH peptide or anti-sclerostin antibody), and the use of an anabolic biophysical modality (LIPUS) have been completed. All the in-vivo micro-CT and X-ray volumes have been analyzed and results reported.

Since the completion of these analyses, programming code has been created for another study that allows us to go back and determine extent of hard callus bridging (percent union value) on a per time point basis. We anticipate using this code to give a better visualization of union in 3D versus the 2D data currently reported. All mechanical testing has been completed and the raw data collected. Data has been reviewed and we are currently finalizing the analyses of the data. Ex-vivo micro-CT callus contents will also be determined and correlated to our mechanical results. Similarly as above, a percent union analysis will be completed on the end-point volumes and correlated to our mechanical results. Histological quantification of callus content for the Aim 3 animals is still underway. Data analyses for these specimens will be the same as those presented for Aim 1. Specifically the quantification of fracture callus tissue will utilize the same histological techniques as in Aim 1. We anticipate finalizing data analyses over the next 6-9 months. The results from this aim will then be compiled for publication with our collaborators at IUPUI (Drs. David Burr and Stuart Warden).

Note change in Element, Risk, Gap to align with IRP Rev C, per JSC HRP (Ed., 5/25/2011)

In its first year, NSBRI MA01604 has determined by micro-CT imaging and histological analyses that closed femoral fractures in HLU rats exhibit a smaller hard callus healing response compared to weight bearing (WB) counterparts. This suggests that healing of closed femoral fractures in HLU rats is altered. Previously in our project NSBRI BL00405, we determined that an extended period of HLU impaired open fibular fracture healing in rats resulting in a non-union rate of >50% after 6-weeks of healing. However the extent of hard callus healing in HLU closed femoral fractures is of greater magnitude as compared to that exhibited by HLU open fibular fractures.

In its second year, NSBRI MA01604 has confirmed the initial findings that closed femoral fractures in HLU rats exhibit substantially smaller hard callus volumes (40-60% smaller than WB ones) even after 10-weeks of healing. Yet, torsion testing assessments of HLU vs. WB hard calluses indicated sound mechanical properties for both HLU and WB calluses, though the HLU calluses were more brittle. Histological assessments at 10-weeks indicate that the content within the HLU calluses is ~40% mineralizing tissue and ~20% soft/fibrous tissue. Assessments of gene expression and tissue alterations for early fracture healing timepoints (1 and 2 weeks post-fracture) correlating to the chrondrogenic phase (soft tissue callus formation) and the beginnings of the endochondral ossification phases (hard tissue callus formation) of fracture healing are complete. This analysis highlights a delay in endochondral ossification due to lagging chondrocyte hypertrophy, in effect delaying subsequent steps of the healing process such as angiogenic vessel infiltration and mineral deposition. Safranin O histological staining results of proteoglycan deposition within the fracture callus from WB and HLU rats suggested similar amounts of hyaline cartilage tissue in each test group. These histological findings are in agreement with those of gene expression findings whereby aggrecan mRNA levels were similar between groups at 1- and 2-weeks post-fracture. This prolonged chondrocyte maturation step exemplified by delayed hypertrophy, reduced osteo-inductive factor expression, and reduced pro-angiogenic factor expression likely leads to a postponement of the requisite vascularization of HLU callus tissue and its subsequent mineralization. While after a full 10 weeks of healing, it is apparent that HLU fractures heal, the data also indicate that the healing process itself may be altered as compared to fractures from WB rats. In fact, fractures from HLU rats are mechanically sound compared to fractures from WB rats. Yet the explanation for this adaptive healing response in HLU callus is not identified currently.

In its third year, NSBRI MA01604 has utilized pharmaceuticals and biophysical stimulation in attempts to augment fracture healing in rat femora. Both WB and HLU rats were given intermittent parathyroid hormone (PTH) injections to stimulate fracture healing and fractures were monitored longitudinally by micro-CT at multiple post-fracture time points. Analysis of this data is ongoing. Low intensity pulsed ultrasound (LIPUS) was also utilized to augment fracture healing in HLU rats. Preliminary findings indicate that fracture callus bridging occurs more rapidly in response to LIPUS than to sham treated femoral fractures. In addition to these findings, further analysis of fracture callus after 10 weeks of non-pharmaceutical or -biophysical stimulated healing by histomorphometrics confirms micro-CT data indicating that total callus volumes are decreased in HLU fractures and that HLU callus tissue contains reduced amounts of cartilagenous and fibrous tissues. Use of anti-sclerostin as a pharmaceutical method to augment healing has not been completed as yet due to delays in obtaining the antibody. We anticipate continuing this study as part of year 4 work.

NASA ARC Aim 2: Tissue histology within the early phases of fracture healing was evaluated. This study included 4 groups, HLU & normal WB at 7- or 14 days post-fracture. Tissue sections from formalin fixed, TCA decalcified, paraffin embedded femora were assessed histologically by safrannin O/fast green. Callus volumes and hyaline cartilage content were quantified.

IUPUI Aim 1: Hard tissue histology and histomorphometry of femoral fracture callus tissue following a ten week healing period will be completed in June 2011. Analysis of fracture callus tissue utilizes toludine blue and von Kossa/McNeal stains to visualize cartilage and mineralized tissue, respectively. Aim 3: One complete trial utilizing Low Intesity Pulsed UltraSound (LIPUS) as a modality to augment fracture healing in WB and HLU rats (n=12 per group) has been completed. A second trial was completed to investigate aspects of prerodding duration on fracture site stabilization. Groups were as follows: 1) fracture 4 weeks following prerodding (n=8), 2) fracture two weeks following prerodding (n=8), 3) fracture immediately following rodding, 4) rodded control (rodded for 4 weeks, no fracture; n=8), 5) cage control (no rod, no fracture; n=5). A third trial consisting of 20 animals with a healing time of 12 weeks is underway. Quantification of callus bridging for the trial 1 will be completed in June 2011.

U of W Aim 1: The method for creating Finite Element (FE) mesh models to describe mechanical properties of healing femora has been streamlined. The FE model will be validated against specimens that have undergone torsional testing to enable model use in future studies to assess mechanical integrity without having to implement destructive testing.

In its first year, NSBRI MA01604 has determined visually using 3D micro-CT imaging that closed femoral fractures in HLU rats exhibit a smaller hard callus healing response when compared to weight bearing (WB) counterparts. In addition, this project has determined that HLU results in the regression of major blood vessels within the hind limb within the initial 2-weeks of HLU. Associated with this vessel regression was a noticeable drop in blood flow rates within the femoral artery. Altogether, these findings suggest that healing of closed femoral fractures in HLU rats is altered as compared to WB healing. The extent of HLU closed femoral hard callus healing is of greater magnitude as compared to HLU open fibular fractures.

In its second year, NSBRI MA01604 has confirmed the initial findings that closed femoral fractures in HLU rats exhibit substantially smaller hard callus volumes (40-60% smaller than WB ones) even after 10-weeks of healing. Yet, torsion testing assessments of HLU vs. WB hard calluses after 10-weeks of healing indicated sound mechanical properties for both HLU and WB calluses, though the HLU calluses were substantially more brittle than their WB counterparts. Thus, closed femoral fracture healing is not impaired and these findings are in stark contrast to those found for HLU open fibular fractures. Assessments for early fracture healing gene expression levels (osteogenic & angiogenic factors, and osteoprogenitor cell numbers) and callus tissue histology in the closed femoral fracture model in HLU versus WB adult rats are ongoing and final outcomes are expected in June 2010.

The research impact of our findings from MA01604 (and those from our prior award BL00405) for Earth based medical practice would suggest that an extended period of unloading and a cephalic fluid shift out of normally weight bearing lower extremity bones may manifest a delayed or an impaired bone healing response. This information may have relevance towards a better understanding of the underlying causes of impaired bone healing in patients experiencing paralysis, chronic immobility or extended bed rest. Previous data obtained from our prior award period suggested that treatments with bone anabolic therapies seem to partially counteract the impairment of bone healing under simulated spaceflight conditions. Our current award will explore additional potential countermeasures in the third year and may also offer potential treatments for augmenting bone healing in Earth-bound, non-weight bearing patients.

(1) 34 WB femora contained desired mid-femoral diaphyseal breaks of simple transverse or bending wedge patterns. 31 HLU femora contained desired mid-femoral diaphyseal breaks of simple transverse or bending wedge patterns. All others failed to fracture, were not localized to the mid-femoral region, or were of incorrect fracture pattern (fragmented or oblique). All unbroken femora will be utilized as controls for any gene expression changes due to medullary reaming and soft tissue injuries associated with the blunt impact of the 3-point bend device. (2) All callus tissues from 1-wk & 2-wk femoral fracture healing have been harvested and snap-frozen in liquid nitrogen. RNA harvest protocol from callus tissue has been optimized to increase the yield & purity of RNA harvested. (3) Quantitative PCR primer sets were redesigned to increase the specificity of amplification, and now span intron:exon junctions with at least one primer from each pair spanning an exon:exon junction. Efficacy of these primers is currently being assessed utilizing a real-time quantitative PCR instrument (ABI 7500). Gene expression analyses to be completed in June 2010.

NASA Ames Research Center (Aim 2)

(1) NASA-ARC arm of MA01604 aims to measure & evaluate the tissue histology within early phases of fracture healing. (2) Study included 4 groups, HLU and normal weight-bearing - 7 days and 14 days post-fracture. Animals (total 79 rats) were received, acclimated to cages and placed in HLU (standardized NASA analog developed by E. Holton) or WB groups. (3) Four weeks after initial HLU, animals underwent bilateral femoral fracture protocol. High resolution x-ray images of the femoral area were taken to verify complete fracture, confirm pin placement, & identify location of fracture. (4) All procedures were performed at ARC with the support of a team from the CCF Midura lab. This ensured that procedures were performed the same way at ARC as normally done at CCF. Also, this approach allowed us to complete the entire study in one experimental run, with significant savings of time and resources. (5) At 7- & 14-days post fracture, animals were euthanized. Blood was collected in heparinized tubes from all animals, including some baseline rats that were part of the original cohort received from the vendor, but housed in standard cages throughout the study. Plasma collected from the animals was stored for future analysis of stress markers. (6) Femora were collected and fixed for histology, and then sent to CCF for micro-CT imaging. Femoral samples were scanned and then decalcified at CCF prior to return to NASA-ARC. (7) Tissue embedding and staining procedures were established. Embedding, sectioning and staining of samples is currently in progress. Histomorphometry will be performed to measure the callus tissue composition, and completed in June 2010. Additional sections will be returned to CCF for immunohistological analysis.

Our objectives are: (1) Determine the scope and extent of femoral fracture healing impairment, (2)Determine the underlying biological causes of the impairment, (3) Develop countermeasures to prevent fracture healing impairment, and (4) Determine whether current Earth-based clinical procedures will reverse severely delayed fracture healing situations resulting from hypogravity. HLU rats will undergo closed femoral fractures and healing will be assessed using (a) micro-CT bone imaging to evaluate hard callus structure, (b) hard callus strength via torsion testing, (c) callus tissue composition using histomorphometry, (d) colony forming unit assessments of marrow-derived osteoprogenitor cell numbers, and (e) measurements of osteoinductive, chondrogenic and angiogenic factor expression during early healing periods.

In its first year, NSBRI MA01604 has determined visually using 3D micro-CT imaging that closed femoral fractures in HLU rats exhibit a delayed healing response when compared to weight bearing (WB) counterparts. In addition, this project has determined that HLU results in the regression of major blood vessels within the hind limb within the initial 2-weeks of HLU. Associated with this vessel regression was a noticeable drop in blood flow rates within the femoral artery. Altogether, these findings suggest healing of closed femoral fractures in HLU rats is impaired, though not to the same magnitude as fibular fractures.

The research impact of our findings from MA01604 (and those from our prior award BL00405) for Earth based medical practice would suggest that an extended period of unloading of normally weight bearing bones should manifest an impaired bone healing response. This information may have relevance towards a better understanding of the underlying causes of impaired bone healing in patients experiencing paralysis, chronic immobility or extended bed rest. Previous data obtained from our prior award period suggested that treatments with bone anabolic therapies seem to partially counteract the impairment of bone healing under a non-weight bearing situation. Our current award will explore this potential countermeasure in the third year and may also offer potential treatments for augmenting bone healing in Earth-bound, non-weight bearing patients.

2) Re-designed and fabricated two 3-point bending devices adapted from the original design published by Bonnarens and Einhorn. One of these devices will stay at the Cleveland Clinic throughout the duration of the 4-year project, while the second one has been shipped to NASA-Ames Research Center for the completion of their subcontract work. The re-design of these devices has resulted in a more consistent generation of closed femoral fractures having a simple transverse pattern within the mid-diaphysis.

3) Design of a threaded titanium rod that (1) ensures that there is no rod slippage or backing out of rods from the femur post surgery and (2) allows for clear micro-CT images (no beam hardening as occurs with stainless steel rods) that are used to determine the hard callus parameters. Thus, we have adapted the Bonnarens & Einhorn closed fracture procedure to be compatible with micro-CT imaging.

4) Software modification for hard callus parameter analyses is currently underway. Existing programs are being modified to account for the titanium rod in the analyses thereby extracting 3-D bone volumes from both in vivo and ex vivo micro-CT imaging.

5) RT-PCR protocol development for the harvest of fracture callus. Primers for gene expression analysis of factors associated with osteogenesis, chondrogenesis, angiogenesis, and inflammation have been identified and specificity checked. Optimal PCR conditions for each primer pair are currently being determined for use in real-time quantitative PCR (Q-PCR).

6) Site visits have been made to both Indiana University (May-June 2008) and NASA-Ames Research Center (April-May 2009). In this first year with Indiana University, standardization of specimen transfer for histomorphometric analyses has been developed. Training of the NASA-Ames personnel and transfer of the standard protocols that will be utilized by all sites has been completed and studies are expected to start mid-summer of this year. Technical support will continue to be provided by personnel at the Cleveland Clinic as needed throughout the second year of this grant.

Objectives

1. Determine the scope and extent of femoral fracture healing impairment.

2. Determine the underlying biological causes of the impairment.

3. Develop countermeasures to prevent fracture healing impairment.

4. Determine whether current Earth-based clinical procedures will reverse severely delayed fracture healing situations resulting from hypogravity.

Tail-suspended rats will undergo closed femoral fractures and healing will be assessed using: micro-CT bone imaging to evaluate hard callus structure; hard callus strength via torsion testing; callus tissue composition using histomorphometry; colony forming unit assessments of marrow-derived osteoprogenitor cell numbers; and measurements of osteoinductive and angiogenic factor expression during early healing periods.