Liisa T. Kuhn

Shape and size of isolated bone mineralites measured using atomic force microscopy

The inorganic phase of bone is comprised primarily of very small mineralites. The size and shape of these mineralites play fundamental roles in maintaining ionic homeostasis and in the biomechanical function of bone. Using atomic force microscopy, we have obtained direct three‐dimensional visual evidence of the size and shape of native protein‐free mineralites isolated from mature bovine bone. Approximately 98% of the mineralites are less than 2 nm thick displaying a plate‐like habit. Distributions of both thickness and width show single peaks. The distribution of lengths may be multimodal with distinct peaks separated by ∼6 nm. Application of our results is expected to be of use in the design of novel orthopaedic biomaterials. In addition, they provide more accurate inputs to molecular‐scale models aimed at predicting the physiological and mechanical behavior of bone.

Size and shape of mineralites in young bovine bone measured by atomic force microscopy

Atomic force microscopy (AFM) was used to obtain three-dimensional images of isolated mineralites extracted from young postnatal bovine bone. The mean mineralite size is 9 nm × 6 nm × 2.0 nm, significantly shorter and thicker than the mineralites of mature bovine bone measured by the same technique. Mineralites of the young postnatal bone can be accommodated within the hole zone regions of a quasi-hexagonally packed collagen fibril in the fashion described by Hodge [9] in which laterally adjacent hole zone regions form continuous “channels” across the diameter of a fibril for a distance of at least 10 nm. Deposition of mineralites of the size noted above in this void volume of the fibrils would result in little or no distortion of the collagen molecules or supramolecular structure of the collagen fibril. The new AFM data supporting this claim is consistent with findings obtained by electron microscopy and low-angle x-ray and neutron diffraction that mineralites formed within collagen fibrils during initial stages of calcification occur within the hole zone region. However, the deposition of additional mineralites in the intermolecular spaces between collagen molecules in the overlap region of the fibrils would significantly distort the fibrils since the space available between adjacent molecules is considerably less than even the smallest dimension of the mineralites.

One-step derivation of mesenchymal stem cell (MSC)-like cells from human pluripotent stem cells on a fibrillar collagen coating.

Controlled differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) into cells that resemble adult mesenchymal stem cells (MSCs) is an attractive approach to obtain a readily available source of progenitor cells for tissue engineering. The present study reports a new method to rapidly derive MSC-like cells from hESCs and hiPSCs, in one step, based on culturing the cells on thin, fibrillar, type I collagen coatings that mimic the structure of physiological collagen. Human H9 ESCs and HDFa-YK26 iPSCs were singly dissociated in the presence of ROCK inhibitor Y-27632, plated onto fibrillar collagen coated plates and cultured in alpha minimum essential medium (alpha-MEM) supplemented with 10% fetal bovine serum, 50 uM magnesium L-ascorbic acid phosphate and 100 nM dexamethasone. While fewer cells attached on the collagen surface initially than standard tissue culture plastic, after culturing for 10 days, resilient colonies of homogenous spindle-shaped cells were obtained. Flow cytometric analysis showed that a high percentage of the derived cells expressed typical MSC surface markers including CD73, CD90, CD105, CD146 and CD166 and were negative as expected for hematopoietic markers CD34 and CD45. The MSC-like cells derived from pluripotent cells were successfully differentiated in vitro into three different lineages: osteogenic, chondrogenic, and adipogenic. Both H9 hES and YK26 iPS cells displayed similar morphological changes during the derivation process and yielded MSC-like cells with similar properties. In conclusion, this study demonstrates that bioimimetic, fibrillar, type I collagen coatings applied to cell culture plates can be used to guide a rapid, efficient derivation of MSC-like cells from both human ES and iPS cells.

Design and characterization of calcium phosphate ceramic scaffolds for bone tissue engineering.

OBJECTIVES Our goal is to review design strategies for the fabrication of calcium phosphate ceramic scaffolds (CPS), in light of their transient role in bone tissue engineering and associated requirements for effective bone regeneration. METHODS We examine the various design options available to meet mechanical and biological requirements of CPS and later focus on the importance of proper characterization of CPS in terms of architecture, mechanical properties and time-sensitive properties such as biodegradability. Finally, relationships between in vitro versus in vivo testing are addressed, with an attempt to highlight reliable performance predictors. RESULTS A combinatory design strategy should be used with CPS, taking into consideration 3D architecture, adequate surface chemistry and topography, all of which are needed to promote bone formation. CPS represent the media of choice for delivery of osteogenic factors and anti-infectives. Non-osteoblast mediated mineral deposition can confound in vitro osteogenesis testing of CPS and therefore the expression of a variety of proteins or genes including collagen type I, bone sialoprotein and osteocalcin should be confirmed in addition to increased mineral content. CONCLUSIONS CPS are a superior scaffold material for bone regeneration because they actively promote osteogenesis. Biodegradability of CPS via calcium and phosphate release represents a unique asset. Structural control of CPS at the macro, micro and nanoscale and their combination with cells and polymeric materials is likely to lead to significant developments in bone tissue engineering.

Structure, Composition, and Maturation of Newly Deposited Calcium-Phosphate Crystals in Chicken Osteoblast Cell Cultures

Characterization of the very early calcium phosphate (CaP) crystals deposited in bone or in osteoblast cell cultures has been hampered by the overwhelming presence of organic matrix components and cells that obscure spectral analyses. We have overcome this problem using isolated protein‐free crystals and have obtained new data including31P nuclear magnetic resonance (NMR) spectra for the first time from mineral crystals deposited during osteoblast calcification in culture. Crystals were isolated from cultures at two time points: (a) at first calcium accumulation (day 8–10) and (b) after 60 days of culture, to assess maturational changes. The analyses show that the chemical composition overall and short range order of the early and mature crystals are characteristic of the apatite crystals found in young embryonic chick bone in vivo. No mineral phase other than apatite was detected by any of the methods used.31P NMR spectroscopy identified the HPO4 groups as those present in bone apatite. Similar to bone apatites, no OH groups were detected by Fourier transform infrared (FTIR) spectroscopy. The temporal maturational changes in composition and structure of the mineral phase were difficult to assess because of the continuous deposition of crystals throughout culturing. The pathway of the maturational changes observed were similar to those occurring in chick bone in vivo and synthetic apatite crystals in vitro although to a much smaller extent.

Osteogenetic Properties of Electrospun Nanofibrous PCL Scaffolds Equipped With Chitosan-Based Nanoreservoirs of Growth Factors

Bioactive implants intended for rapid, robust, and durable bone tissue regeneration are presented. The implants are based on nanofibrous 3D-scaffolds of bioresorbable poly-ϵ-caprolactone mimicking the fibrillar architecture of bone matrix. Layer-by-layer nanoimmobilization of the growth factor BMP-2 in association with chitosan (CHI) or poly-L-lysine over the nanofibers is described. The osteogenetic potential of the scaffolds coated with layers of CHI and BMP-2 is demonstrated in vitro, and in vivo in mouse calvaria, through enhanced osteopontin gene expression and calcium phosphate biomineralization. The therapeutic strategy described here contributes to the field of regenerative medicine, as it proposes a route toward efficient repair of bone defects at reduced risk and cost level.

Fibroblast Growth Factor-2 and Bone Morphogenetic Protein-2 Have a Synergistic Stimulatory Effect on Bone Formation in Cell Cultures From Elderly Mouse and Human Bone

Combined regimens of fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2) were investigated to stimulate osteogenic differentiation. In young mouse calvaria-derived cells, FGF-2 (0.16ng/mL) in combination with BMP-2 (50ng/mL) did not enhance mineralization, but in old mouse cells it resulted in more mineralization than BMP-2 alone. In young long bone mouse cultures, FGF-2 enhanced mineralization relative to BMP-2 alone, but in old cultures, lower dose of FGF-2 (0.016ng/mL) was necessary. In neonatal mouse calvarial cells, sequential delivery of low-dose FGF-2 and low-dose BMP-2 (5ng/mL) was more stimulatory than co-delivery. In young human cultures, 0.016ng/mL of FGF-2 did not enhance mineralization, in combination with 5ng/mL of BMP-2, but in older cultures, codelivery of FGF-2 and BMP-2 was superior to BMP-2 alone. In conclusion, BMP-2 treatment alone was sufficient for maximal mineralization in young osteoblast cultures. However, coadministration of FGF-2 and BMP-2 increases mineralization more than BMP-2 alone in cultures from old and young mouse long bones and old humans but not in young mouse calvarial cultures.

Imaging tumor hypoxia by near-infrared fluorescence tomography

We have developed a novel nitroimidazole indocyanine dye conjugate for tumor-targeted hypoxia fluorescence tomography. The hypoxia probe has been evaluated in vitro using tumor cell lines and in vivo with tumor targeting in mice. The in vitro cell studies were performed to assess fluorescence labeling differences between hypoxia and normoxia conditions. When treated with the hypoxia probe, a fluorescence emission ratio of 2.5-fold was found between the cells incubated under hypoxia compared to the cells in normoxia condition. Hypoxia specificity was also confirmed by comparing the cells treated with indocyanine dye alone. In vivo tumor targeting in mice showed that the fluorescence signals measured at the tumor site were twice those at the normal site after 150 min post-injection of the hypoxia probe. On the other hand, the fluorescence signals measured after injection of indocyanine dye were the same at tumor and normal sites. In vivo fluorescence tomography images of mice injected with the hypoxia probe showed that the probe remained for more than 5 to 7 h in the tumors, however, the images of mice injected with indocyanine only dye confirmed that the unbound dye washed out in less than 3 h. These findings are supported with fluorescence images of histological sections of tumor samples using a Li-COR scanner and immunohistochemistry technique for tumor hypoxia.

An evaluation of BMP-2 delivery from scaffolds with miniaturized dental implants in a novel rat mandible model

The purpose of this study was three-fold: (a) to develop a new small animal model to evaluate dental implant systems that recapitulates aspects of the challenging intraoral environment, (b) screen several scaffolds for in vivo bone forming efficacy when used to deliver non-glycosylated bone morphogenetic protein-2 (BMP-2) together with a miniaturized titanium (Ti) dental implant, and (c) identify correlations between in vitro BMP-2 release rates and in vivo results. The scaffolds tested were: (1) collagen-hydroxyapatite composite (Col/HA), (2) polyethylene glycol hydrogel (PEG-hydrogel), and (3) Col/HA infused with PEG-hydrogel (Col/HA/PEG-hydrogel). BMP-2 delivery directly from the Ti implants rather than from the scaffolds was also tested. MicroCT analyses at 4 weeks showed that the maximum volume and height of new bone occurred when BMP-2 (10 μg) was delivered from the Col/HA/PEG-hydrogel scaffolds. BMP-2 delivery from the Ti implant was not as effective as from the scaffolds. While in vitro BMP-2 release was highest for the PEG-hydrogel, the scaffold most successful in vivo was the Col/HA/PEG-hydrogel scaffold because it had the necessary mechanical strength to perform well in the mandibular bone environment. The in vitro release studies suggested a threshold dose of 5 μg which was borne out by the in vivo dose response studies.

Implant system for guiding a new layer of bone. Computed microtomography and histomorphometric analysis in the rabbit mandible.

OBJECTIVE To prove the concept that an implant system with osteoconductive surface characteristics and an osteoinductive scaffold material has the capacity to guide vertical supracrestal bone growth in a rabbit mandible onlay model. MATERIAL AND METHODS Thirteen adult white New Zealand rabbits each received custom-designed dental implants. All implants had sandblasted, acid-etched (SLA) surfaces, with the coronal aspect (3 mm) of each implant was left outside the lateral aspect of posterior mandibular bone, but covered by periosteum, muscle, subcutaneous tissue, and skin. Bone formation around implants placed adjacent to osteoinductive demineralized bone matrix (DBM) scaffolds were compared with contralateral implants without scaffolds in six rabbits using micro-CT imaging. Bone formation around implants with scaffolds from seven additional rabbits was measured using both micro-CT imaging and quantitative histology. RESULTS At 8 weeks, new supracrestal bone was seen adjacent to all implants placed with DBM and two implants without DBM. The mean supracrestal bone heights achieved for implants with and without DBM scaffolds as measured by micro-CT was 2.1+/-0.9 and 0.8+/-0.9 mm, respectively (P=0.008). Histomorphometric analysis illustrated that supracrestal bone-to-implant contact for implants with DBM scaffolds was 58.1+/-14% and that mean supracrestal bone height was 2.4+/-0.6 mm. CONCLUSIONS Successful implant-guided supracrestal osteogenesis has been demonstrated in a rabbit model with the combined use of osteoconductive implant surfaces, an osteoinductive scaffold, and a device that prevents soft tissue downgrowth and provides scaffold stabilization.