Involvement of calvarial stem cells in healing: A regional analysis of large cranial defects

Abstract

Large craniofacial defects present a substantial clinical challenge that often requires the use of osteoconductive matrices and osteoinductive cues (i.e., bone morphogenetic proteins [BMP2]) to augment healing. While these methods have improved clinical outcomes, a better understanding of how the osteogenic fronts surrounding the defect, the underlying dura mater, and the cranial suture area contribute to healing may lead to more targeted therapies to enhance bone regeneration. We hypothesized that healing within a large bone defect will be precipitated from cells within the remaining or available suture mesenchyme abutting the edges of a murine critical sized defect. To investigate this hypothesis, 39 adult, wild-type mice were randomly arranged into groups (9 or 10 per group) by time (4 and 8 weeks) and treatment (control, acellular collagen sponge alone, or acellular collagen sponge loaded with a clinically relevant scaled dosage of BMP2). The skulls were then subjected to microcomputed tomography and histological analysis to assess bone regeneration in regions of interest within the defect area. A regional assessment of healing indicated that BMP2 drives greater healing than control and that healing emanates from the surgical margin, particularly from the margin associated with undisrupted suture mesenchyme. Though BMP2 treatment drove an increase in cell presence within the healing defect, there was no regional orientation of craniofacial stem cells or vascularity. Overall, these data reinforce that osteoconductive matrices in conjunction with osteoinductive peptides result in better healing of large calvarial defects. This healing is characterized as emanating from the surgical margin where there is an abundant supply of vasculature and progenitor cells.

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

“…This outcome agrees with previous studies that found that the osteoinductive BMP2 along with an osteoconductive matrix such as ACS precipitates the greatest amount of bone growth [16, 25, 27, 31, 32]. Though we hoped that as in previous studies Talymed would precipitate denser bone growth, this did not occur when it was used with the ACS scaffold wounds [8, 9]. However, interestingly our lowest dose of BMP2 (medium) did provide the greatest amount of healing with the least amount of ectopic bone.…”

“…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.…”

“…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.…”

“…Despite the clinical efficacy of BMP2 in bone regeneration, adverse clinical events such as extreme inflammation and edema, as well as poor quality bone regenerate and uncontrolled ectopic bone formation necessitate optimization of the application of this highly effective osteogenic factor [12–15]. The current United States-Food and Drug Administration approved scaffold for BMP2 delivery is absorbable collagen sponge (ACS; INFUSE Bone Graft, Medtronic TM), however use of this scaffold peptide combination has been linked to aberrant inflammatory responses precipitating poor quality bone and ectopic bone growth [8, 9, 16–19]. …”

Sub‐clinical dose of bone morphogenetic protein‐2 does not precipitate rampant, sustained inflammatory response in bone wound healing

“…As such, a comprehensive evaluation of the effect of rhBMP‐2 on cytokine signaling and inflammation is necessary. The present study aimed to investigate alterations in the inflammatory cascade following the application of a sub‐clinical dose of rhBMP‐2, which has been shown to produce substantial bone fill without ectopic bone formation in preclinical models of bone wounds . We hypothesized based on previous work with rhBMP‐2 in preclinical models that did not precipitate negative side effects of rhBMP2 treatment, that inflammatory cascades would not be dramatically affected following application of this rhBMP‐2 dose, evidenced by normal inflammatory cytokine signaling and un‐altered macrophage polarization.…”