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. 2015 Mar;21(5-6):970-81.
doi: 10.1089/ten.TEA.2013.0789. Epub 2014 Dec 16.

Osseointegrative properties of electrospun hydroxyapatite-containing nanofibrous chitosan scaffolds

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Osseointegrative properties of electrospun hydroxyapatite-containing nanofibrous chitosan scaffolds

Michael E Frohbergh et al. Tissue Eng Part A. 2015 Mar.

Abstract

Our long-term goal is to develop smart biomaterials that can facilitate regeneration of critical-size craniofacial lesions. In this study, we tested the hypothesis that biomimetic scaffolds electrospun from chitosan (CTS) will promote tissue repair and regeneration in a critical size calvarial defect. To test this hypothesis, we first compared in vitro ability of electrospun CTS scaffolds crosslinked with genipin (CTS-GP) to those of mineralized CTS-GP scaffolds containing hydroxyapatite (CTS-HA-GP), by assessing proliferation/metabolic activity and alkaline phosphatase (ALP) levels of murine mesenchymal stem cells (mMSCs). The cells' metabolic activity exhibited a biphasic behavior, indicative of initial proliferation followed by subsequent differentiation for all scaffolds. ALP activity of mMSCs, a surrogate measure of osteogenic differentiation, increased over time in culture. After 3 weeks in maintenance medium, ALP activity of mMSCs seeded onto CTS-HA-GP scaffolds was approximately two times higher than that of cells cultured on CTS-GP scaffolds. The mineralized CTS-HA-GP scaffolds were also osseointegrative in vivo, as inferred from the enhanced bone regeneration in a murine model of critical size calvarial defects. Tissue regeneration was evaluated over a 3 month period by microCT and histology (Hematoxylin and Eosin and Masson's Trichrome). Treatment of the lesions with CTS-HA-GP scaffolds induced a 38% increase in the area of de novo generated mineralized tissue area after 3 months, whereas CTS-GP scaffolds only led to a 10% increase. Preseeding with mMSCs significantly enhanced the regenerative capacity of CTS-GP scaffolds (by ∼3-fold), to 35% increase in mineralized tissue area after 3 months. CTS-HA-GP scaffolds preseeded with mMSCs yielded 45% new mineralized tissue formation in the defects. We conclude that the presence of HA in the CTS-GP scaffolds significantly enhances their osseointegrative capacity and that mineralized chitosan-based scaffolds crosslinked with genipin may represent a unique biomaterial with possible clinical relevance for the repair of critical calvarial bone defects.

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Figures

<b>FIG. 1.</b>
FIG. 1.
Morphology of electrospun CTS-HA-GP scaffolds. Scanning electron micrographs of electrospun mineralized scaffolds (original magnification: 1000×) indicate a network of randomly oriented, beadless fibers. The insert, taken at higher magnification (5000×), shows the presence of hydroxyapatite (HA) nanoparticles studding the surface of the electrospun fibers (white arrows). Scale bars are 100 μm (main figure) and 200 nm (insert), respectively. CTS, chitosan; GP, genipin.
<b>FIG. 2.</b>
FIG. 2.
Morphology of murine mesenchymal stem cells (mMSCs) seeded onto chitosan (CTS) scaffolds. (A–D) Cells cultured on CTS-GP scaffolds at 1 week (A) and 3 weeks (C) and CTS-HA-GP scaffolds at 1 week (B) and 3 weeks (D) were fixed and stained with DAPI (nuclei, blue) and Phalloidin (F-actin, green). The cultures quickly reached and then maintained confluence throughout the study. (E, F) Three-dimensional renderings of the scaffolds demonstrated a three-fold increase (p>0.05) in thickness of the cellular layer between 1 week (E) at 8 μm and 2 weeks (F) at 24 μm. Scale bars for all panels=50 μm, original magnification for all images: 10×. Color images available online at www.liebertpub.com/tea
<b>FIG. 3.</b>
FIG. 3.
The alamarBlue™ fluorescence and alkaline phosphatase (ALP) activity of mMSCs seeded onto the various scaffolds. Murine MSCs were seeded onto the various scaffolds in either maintenance medium (Iscove's modified Dulbecco's medium) or osteogenic medium (OGM). AB fluorescence (for metabolic activity) and ALP activity (for osteogenic differentiation) were measured at the times indicated (for details see Materials and Methods section). All data are presented as means±standard deviations. Sample size was n=3 (in triplicate) with *p<0.05 and **p<0.01 compared with the previous time points of the same group and ++p<0.01 compared with the CTS-GP control group at the same time point. (A) Continual measurement of alamarBlue™ fluorescence revealed that by day 10 the metabolic activity of mMSCs decreased on all scaffolds, and plateaued until day 21 (data are expressed as arbitrary fluorescence units). (B) ALP activity normalized to mMSCs cultured in parallel on tissue culture polystyrene (TCP), increased in the presence of HA and OGM when compared with CTS-GP cultures alone.
<b>FIG. 4.</b>
FIG. 4.
Reconstructed microCT images of calvarial defect treatments. Formation of new mineralized tissue at 3 months. The size and location of the original lesions are depicted by the orange circular regions of interest. Representative μCT images are shown. (A) CTS-GP scaffolds without cells; (B): CTS-GP-HA scaffolds without cells; (C): CTS-GP scaffolds with cells; (D): CTS-GP-HA scaffolds with cells. Scale bar=5 mm. Color images available online at www.liebertpub.com/tea
<b>FIG. 5.</b>
FIG. 5.
Bone regeneration in critical size calvarial defects. The percentage (%) of defect closure in the presence of the scaffolds was normalized to the size of the contralateral nontreated control defects in each sample at every time point. No or minimal healing was observed in untreated defects at all times. Nonmineralized scaffolds were effective after 3 months, but only when preseeded with mMSCs. Most pronounced healing over time was observed in lesions treated with scaffolds preseeded with mMSCs. However, at 3 months there was no statistically significant difference in the percentage of wound closure in lesions treated with mineralized scaffold without or with cells. For details see text. Data are presented as means±standard deviations. The sample size (number of individual mice) was n=4 for all measurements, with *p<0.05 and **p<0.01 compared with nonmineralized scaffolds.
<b>FIG. 6.</b>
FIG. 6.
Histology of healing of critical size calvarial defects 3 months after injury. Nontreated defects show no or minimal wound healing and coverage of the lesion with a thin fibrous layer that is comprised of collagen. (A) Hematoxylin and Eosin (H&E) staining, (B): Masson's Trichrome stain (MTS), original magnification of (A, B) is 40×. H&E (C) and Masson's Trichrome (D) staining of defects treated with CTS-HA-GP scaffolds without mMSCs show new bone formation (red), collagen deposition (blue), and significant scaffold interaction. Original magnification 40×, inserts are 100×. H&E (E) and Masson's Trichrome (F) staining of defects treated with CTS-HA-GP scaffolds pressed with mMSCs indicates very similar results to those scaffolds without cells, but does appear to have enhanced tissue growth and new bone formation along the periphery of the scaffold (brown). Higher magnification of a defect treated with CTS-HA-GP scaffolds seeded with mMSCs reveals endochondral-like tissue formation along the host–scaffold interface (F). Original magnification 40×, inserts are 100×. Magnification for (C–F) are 100×. For details, see text. Scale bars: (A, B): 250 μm; (C–F): 100 μm. Color images available online at www.liebertpub.com/tea

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