Despite starting with the statistically smallest defects, the bone gaps treated only with PPCN-g scaffold did not statistically change in size when evaluated eight weeks postoperatively (p = 0.72). Conversely, both animal groups treated with iCALs showed significant reductions in defect size compared to baseline (week 0) after 12 weeks of follow-up (AdBMP9-treated: p = 0.0025; AdGFP-treated: p = 0.0042). A significant difference between test groups was detected by one-way ANOVA by 6 weeks of follow-up (p = 0.0003) ( Fig 3A–3C ). The PPCN-g/AdBMP9-treated defects failed to exhibit greater bone regeneration compared to the control (PPCN-GFP) group at 6 weeks postoperatively (p = 0.11); however, by 12 weeks, significantly greater bone regeneration was apparent in the BMP9 treatment group. (p = 0.027) ( Fig 3E and 3F ). Interestingly, between 6 and 12 weeks, both iCAL-treated groups showed an increase in average defect size, the extent of which was more than twice as great in the PPCN-g/AdGFP-treated group (0.96 mm 3 increase) compared with the PPCN-g/AdBMP9-treated group (0.41 mm 3 increase). This may be attributed to better bone formation in the BMP group than in the GFP group although exact causes require further investigation.

(A, B, C) Average defect volumes (mm 3 ) were calculated at 24–48 hours, 2, 4, 6, 8, and 12 weeks postoperatively using volumetric reconstructions generated in Amira®. Asterisks indicate a significant (p < 0.05) difference in average defect volume between test groups at the specified time point, as determined by one-way ANOVA. (D, E, F) The change in defect volume over time was used to deduce the percentage of baseline defect volume filled with new bone.

In order to assess the dynamic process of osteogenesis at the repair sites, microCT imaging was performed at 48 hours, 2, 6, 7, 8, and 12 weeks postoperatively ( Fig 2 ). Volumetric reconstructions were generated using Amira® and defect volumes were quantitatively determined. Baseline imaging at 48 hours postoperatively revealed uniform circular defects, with average volumes of 2.99 mm 3 , 3.00 mm 3 , and 2.50 mm 3 for PPCN-g/AdBMP9 ( Fig 3C ), PPCN-g/AdGFP ( Fig 3B ), and PPCN-g alone ( Fig 3A ), respectively. A significant difference between baseline defect volumes of our test groups was detected via one-way ANOVA (p = 0.0049).

In terms of morbidity and mortality, all animals survived the surgery. However, two out of seven mice in the iCAL-BMP9 treatment group died within 24 hours of surgery and one of seven mice in the iCAL-GFP control group died 11 weeks postoperatively. Other complications were technical in nature. During the procedure of a mouse from the iCAL-GFP group, the implant was inadvertently displaced from the defect site prior to solidification and could not be recovered. CT imaging data from an additional mouse in the iCAL-BMP9 group at week 2 showed bone formation in a pattern that suggested lateral displacement of the implant. Data from all of the above mentioned mice were omitted from analysis. All surviving mice progressed well postoperatively, gaining an average 1.2 grams/week and apparently with well-treated pain.

Histologic analysis of tissue microsections harvested 12 weeks post-treatment. Yellow arrows indicate the original defect borders. (A) The defect site of this AdGFP-treated mouse shows incomplete healing; there is some ingrowth of bone, but fibrous tissue fills part of the defect. (B) Conversely, the defect site of an AdBMP9-treated mouse has been completely bridged with new bone. All sections show no trace of PPCN material, indicating complete resorption.

While microCT imaging provides an assessment of the dynamics of the defect repair process, the quality of bony repair at defect sites requires examination by histologic analysis. H & E and trichrome staining of the retrieved samples confirmed incomplete healing in PPCN-g/AdGFP-treated group as the defects were primarily bridged with fibrous tissue ( Fig 4A ) and cartilage ( Fig 5A and 5B ). Conversely, robust bone formation was seen in the PPCN-g/AdBMP9-treated group, with some defects filled in completely with new bone ( Fig 4B ). Trichrome staining of calvarial defect microsections harvested at 12 weeks post-treatment corroborated these findings by showing a higher proportion of mature bone in the PPCN-g/AbBMP9-treated defect sites ( Fig 5C and 5D ) than in those treated with PPCN-g/AdGFP ( Fig 5A and 5B ). In all samples, no trace of PPCN-g material was observed, suggesting that the scaffold was completely degraded, which was further confirmed in the PPCN-g alone group ( S1 Fig ).

Combining BMP9 induction and cell delivery via the PPCN-g scaffold is a promising stem cell-based therapeutic strategy for cranial defect repair

Efficacious bone regeneration requires integration with surrounding tissue, including vascularization, fusion of the implant with autologous bone without fibrous tissue at the bone-implant interface, and eventual complete replacement of the scaffold with new bone [34–36]. In this study we have demonstrated that a novel class of thermoresponsive biomaterial loaded with BMP9-induced calvarial cells is capable of producing bone that successfully integrates with neighboring tissue.

One of the components contributing to this demonstration was the PPCN-g scaffold, which provided a temporary framework for osteoblastic differentiation and osteogenesis. Histology confirms bone formation, and complete degradation of the scaffold. We did not obtain and analyze serial histological samples and therefore cannot thoroughly comment on the degradation kinetics of PPCN-g, but previous in vitro studies have determined that the material undergoes hydrolytic breakdown over a period of several weeks and is resorbed by the body [23]. Grossly, no signs of infection, dehiscence, or scaffold extrusion were observed.

Another component contributing to successful bone regeneration and defect healing was BMP9 and its ability to induce osteoblastic differentiation of iCALs. BMP9 has previously exhibited considerable potency in osteoblastic induction [14], and utilization of adenoviral vector gene therapy permits localization of BMP-secreting cells [15,16]. Trichrome staining of histologic microsections harvested at 12 weeks postoperatively demonstrated bridging of those defects with fibrocartilage in the GFP only treatment group rather than bone in the BMP-9 treatment group. Both microCT and histology results support bone formation only in the PPCN-g + iCALs + AdBMP9-treated defects. BMP9 appears to induce direct osteogenic differentiation, leading to intramembranous ossification without a fibrocartilage intermediate. Interestingly, without BMP-9 stimulation, the Ad-GFP infected iCALs embedded in PPCN-g appear to assume a cartilaginous phenotype upon differentiation (Fig 5A and 5B). This chondroid phenotype is a consistent finding no matter what progenitor cell source we have used in our experimentation—iCALs [15,16], immortalized murine embryonic fibroblasts (iMEFs) [26], immortalized murine adipocytes (iMADs) [27], and may represent a differentiation “default” for cells without BMP9 stimulation in the PPCN microenvironment. Overall, these results reiterate previous findings from our lab that demonstrate the superior ectopic bone quality produced by BMP9-transduced iCALs compared to that produced by GFP-transduced iCALs [15,16].

The novelty of this study is threefold. First, this is the first study to demonstrate Ad-BMP-9 induced osteogenesis is effective in the repair of a critical-sized calvarial defect. Secondly, we have shown that immortalized calvarial progenitor cells can be induced in vivo to repair osseous defects of the skull in the mouse. Thirdly, we have introduced a recently developed thermoresponsive material, PPCN-g in the context of craniofacial reconstruction. The decision to use PPCN-g was made based on two major advantages over traditional solid scaffolds. First, because of its initial liquid phase that subsequently solidifies upon introduction into the defect, PPCN-g spontaneously molds to the shape of even the most complicated defects, which may be beneficial with the complex three-dimensional morphology of the skull and facial bones. Second, liquid-phase PPCN-g has the potential to be delivered through minimally invasive means. This has important implications for reducing scaffold dislodgement and contour irregularities that can arise from imprecise scaffold fit. An additional benefit may be the addition of gelatin to PPCN, which provides cell adhesion sites for osteoconduction. Recent data from our laboratory suggest that the addition of gelatin (0.1%) to PPCN enhances angiogenesis within the material. [26]. Specifically, PPCN-g seeded with BMP9-transduced immortalized mouse embryonic fibroblasts (iMEFs) produced statistically more mature bone and VEGF expression than similarly transduced iMEFs in an in vivo ectopic bone assay [25]. Such an interpenetrating network, in combination with BMP9, may be an effective strategy to produced well-vascularized, mature bone in defects under even more hostile conditions such as radiation or infection.

Although the results presented herein are promising, there are limitations to the study. A major limitation of this study is the small sample size of the three groups. Secondly, there were minor differences in initial cranial defect size, however it is important to note that the treatment group defect size was slightly larger than the control group, which re-emphasizes the potentially important therapeutic role of BMP-9 in critical-sized cranial defect repair. Some may argue that another limitation of this study and its digression from clinical relevance is the use of adenovirus to express BMP-9 in infected progenitor cells. To this end, recent studies have investigated the efficacy of recombinant human BMP-9 in osteogenic differentiation and cranial defect repair [37, 38]. Although these studies reiterate our finding of the superior potency of BMP-9 over other osteogenic BMPs [37], and the ability to regenerate new bone in rat critical size calvarial defects [38], several issues subvert the use of rhBMP9. First, the recombinant form is highly expensive, as it is purified from CHO or HEK 293 cells. Furthermore, to date there is no recombinant form yet to demonstrate osteoactivity from bacterial sources. Therefore, applications to repair large cranial defects in humans, the sole driving force to investigate tissue engineering strategies, using rhBMP9 will be confounded by its prohibitive cost. Additionally, as the biological activity of this recombinant protein is largely unknown, achieving the correct dose-response relationship will be challenging to align bioavailability of the protein with healing time needed for complete reconstruction of a given defect. Along these lines, the representative histology provided in the literature regarding its use in critical sized calvarial defects demonstrate incomplete healing of the treated defects, with chondroid and osseous material present in the regenerate [38].

Despite achieving statistically significant differences in defect filling between all three groups (CI 95%), future studies should include larger sample sizes. Lastly, we propose that subsequent iterations of this study be expanded and scaled to utilize larger animals such as rabbits or pigs.