Optimizing bone wound healing using BMP2 with absorbable collagen sponge and Talymed nanofiber scaffold

Adult (8 week old) male and female C57BL6 mice (n = 80) (Jackson Laboratory, Bar Harbor, ME) were randomized into one of eight surgical groups implanted with ACS alone or ACS overlaid with Talymed (ACS/Talymed) matrix soak loaded with control (sterile H₂O), medium (325 ng), high (542 ng) or scaled clinical (5000 ng) doses of BMP2 (Table 1, Fig. 1). Under sterile conditions, 6 mm biopsy punches of ACS and 5 mm biopsy punches of Talymed were obtained and stored in a 24 well plate lined with sterile gauze. Fifteen minutes prior to implantation, matrices were loaded with BMP2 resuspended in 25 µl sterile water (Infuse, Medtronic, Memphis, TN) [9, 26]. One Small Kit of Infuse Bone Graft (Medtronic) which includes sterile water, sterile BMP2, and sterile absorbable collagen sponge (ACS) was used to complete this entire investigation.

For each specimen, a critical-sized calvarial defect was performed as previously described [8, 9, 26‐28]. Briefly, the mice were anesthetized with isoflurane (Bethlehem, PA, USA) and a midline scalp incision was used to expose and remove the periosteum. A 5-mm round craniectomy defect was created using a slow-speed hand drill. After the application of experimental treatment, the incision was closed with 6 × 0 polypropylene suture (removed 2 weeks later). Animals were weighed and monitored 2 days post-surgery for any signs of pain or distress. Daily monitoring continued until time of sacrifice (4 weeks) when mice were euthanized by asphyxiation using CO₂ gas followed by secondary cervical dislocation. Skulls were collected for downstream analyses. All procedures were carried out with the approval of the Medical University of South Carolina IACUC (AR #3452), in an Association for Assessment and Accreditation of Laboratory Animal Care International accredited facility where all husbandry and related services were provided by the Division of Laboratory Animal Resources. The animals were housed 5 to a cage which contained enrichment material and were allowed access to food and water ad libitum. All procedures and the reporting thereof comply with the Animal Research: Reporting in Vivo Experiments (ARRIVE) guidelines [29].

Collected skulls were removed of soft tissue and bisected from the occipital protuberance to nasale in the transaxial plane. Micro-computed tomography (µCT) images of the defect areas were obtained via ex vivo Skyscan 1176 (Skyscan, Aartlesaar, Belgium) with a 0.5 mm thick aluminum filter at a voltage of 50 kV and current of 500 µA. Data were acquired at an isotropic resolution of 18 µm, rotation step of 0.5°, and 180° rotation. Reconstruction and analyses were performed on the blinded samples using NRecon and CTAn SkyScan software. A global threshold of 100–255 was used. Standard 3D morphometric parameters [30] were determined for the region of interest (ROI; 5.0 mm circle, 51 cuts equaling 918 µm), positioned along the initial surgical margins for all samples. A secondary region of interest encompassing all ectopic bone for each sample was also analyzed for standard morphometric parameters. Representative 3D images were created using CTvol software.

Blinded, representative samples (n = 4) from each group were bisected in the coronal plane through the center of the surgical defect and prepared for paraffin sectioning by fixing in 3.7% formaldehyde for 2 days. Subsequently, samples were decalcified in 0.25 M EDTA at pH 7.4 for 21 days and then washed, dehydrated in graded ethanol (70–100%), cleared in xylene, and embedded in paraffin. Histology was performed on 3, 7 µm sections in the coronal plane at least 40 μm apart per sample for analysis of the defect area. Standard procedures were employed for hematoxylin and eosin, Masson’s trichrome (Thermo Scientific, Waltham, MA USA), and picro sirius red (non-polarized and polarized) staining [8, 9]. Stained sections were photographed using a Motic Inverted Microscope with attached camera (Motic, British Columbia, Canada) and quantified using NIH Image J and Visiopharm (Visiopharm, Broomfield, Colorado) software. For analysis, a region of interest including the surgical margins but excluding ectopic bone growth above the native bone along the surgical plane was isolated. Nuclei within the defects were quantified using color deconvolution and creation of masks to count darkly stained nuclei. Total area of bone (osteoid) was analyzed for Mason’s trichrome, and picro sirius red sections were analyzed for total collagen area (non-polarized), thin immature (green; polarized), intermediate (yellow; polarized), and thick mature (red; polarized) collagen fibers.

Two-way ANOVA Scaffold x Treatment with post hoc Bonferroni analyses were conducted for all comparisons where appropriate. Transformations of the data were performed where needed to correct for violations and meet the assumptions of homogeneity of variance or normality. Non-parametric analyses were utilized where necessary for unavoidable violations of assumptions. Differences were considered significant if p ≤ 0.05. Data are presented as mean ± SEM. A power study for experimentation was calculated based on an α = 0.05, β = 0.8, η > 0.4, necessitating 10 animals per group. *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.