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In Vivo Bone Growth Assessment in Preclinical Studies and Clinical Trials

The significance of in vivo investigation has become increasingly important as bone substitutes have grown in structural and compositional complexity. The diversity of parameters currently expected from osseointegration studies, combined with patient or animal population-based variability in reaction/response to such surrogates, necessitates unrealistically large cohorts for adequate power and ultimate statistical significance using standard ex vivo histological assessment. Unfortunately, such large sample sizes translate to increased animal or per-patient costs and exponentially higher turnaround times for sample evaluation.

Recently, with its ability to provide accurate and reproducible bone metrics less susceptible to observer-based bias, use of imaging analytics has gained significant traction to enable efficient assessment of larger ex vivo sample numbers. While this automated approach provides a solution that addresses throughput issues in large studies, the associated costs stemming from animal housing or patient enrollment to sample prep and processing can significantly impact feasibility. Fortunately, the combination of in vivo investigation with novel but validated image analysis techniques 1) drastically reduces the burden of proof required to account for patient/animal population variability as subjects, each with their own baselines for normalization, are followed longitudinally, 2) provides consistent, accurate metrics that can be compared across time points without corruption by user interaction or subjectivity, 3) eliminates the need for sub-sampling employed to reduce costs, 4) provides quantitative and visual evidence of analysis output metrics for marketing or regulatory submissions and 5) lowers statistical variability to enable reduction in the size of animal/patient cohorts.

Ex vivo Assessment

Traditional Histological Evaluation

Histological sectioning and staining of explanted tissues is the current “gold standard” for assessing bone ingrowth into porous, defect-filling scaffolds, bone remodeling around solid implants or fixation hardware, and fracture healing in both research and regulatory realms. Using high resolution optics, histological evaluation enables both gross ultra-structural/organ-level interrogation and visual sensitivity down to the cellular-level. Furthermore, the availability of numerous stains and antibodies, both fluorescent and bright-field allow delineation of tissue types or presence and localization of specific proteins with submicron precision.

Unfortunately, the valuable information gleaned comes with a significant price tag and lengthy turnaround times. Consider the bone implant shown in Exhibit 1 (A & B). To cross-section and stain the porous scaffold completely would require ~200 slices at 100 um intervals to cover its 2 cm diameter. At a conservative rate of $50/section for histological processing/staining, a single sample would require a $10,000 investment and an optimistic 3-6 month turnaround. Additionally, once the sections are generated, a pathologist or multiple “trained” observers are engaged to grade each section and provide a qualitative score for bone ingrowth, lengthening timelines yet again. Obviously pursuing such an aggressive histological survey is not feasible from the standpoint of either cost or efficiency. Thus, most researchers will drastically reduce the number of sections generated to stay within a specific budget and meet appropriate deadlines.