Christina Møller Andreasen

Bone Formation by Sheep Stem Cells in an Ectopic Mouse Model: Comparison of Adipose and Bone Marrow Derived Cells and Identification of Donor-Derived Bone by Antibody Staining

Background. Scaffolds for bone tissue engineering (BTE) can be loaded with stem and progenitor cells (SPC) from different sources to improve osteogenesis. SPC can be found in bone marrow, adipose tissue, and other tissues. Little is known about osteogenic potential of adipose-derived culture expanded, adherent cells (A-CEAC). This study compares in vivo osteogenic capacity between A-CEAC and bone marrow derived culture expanded, adherent cells (BM-CEAC). Method. A-CEAC and BM-CEAC were isolated from five female sheep and seeded on hydroxyapatite granules prior to subcutaneous implantation in immunodeficient mice. The doses of cells in the implants were 0.5 × 106, 1.0 × 106, or 1.5 × 106 A-CEAC and 0.5 × 106 BM-CEAC, respectively. After eight weeks, bone volume versus total tissue volume (BV/TV) was quantified using histomorphometry. Origin of new bone was assessed using human vimentin (HVIM) antibody staining. Results. BM-CEAC yielded significantly higher BV/TV than any A-CEAC group, and differences between A-CEAC groups were not statistically significant. HVIM antibody stain was successfully used to identify sheep cells in this model. Conclusion. A-CEAC and BM-CEAC were capable of forming bone, and BM-CEAC yielded significantly higher BV/TV than any A-CEAC group. In vitro treatment to enhance osteogenic capacity of A-CEAC is suggested for further research in ovine bone tissue engineering.

Pit- and trench-forming osteoclasts: a distinction that matters

Osteoclasts (OCs) seeded on bone slices either drill round pits or dig long trenches. Whereas pits correspond to intermittent resorption, trenches correspond to continuous and faster resorption and require a distinct assembly of the resorption apparatus. It is unknown whether the distinction between pits and trenches has any biological relevance. Using OCs prepared from different blood donors, we found that female OCs achieved increased resorption mainly through pit formation, whereas male OCs did so through trench formation. Trench formation went along with high collagenolytic activity and high cathepsin K (CatK) expression, thereby allowing deeper demineralization. A specific CatK inhibitor abrogated the generation of trenches, while still allowing the generation of pits. OCs obtained from bone marrow were more prone to generate trenches than those obtained from blood. Scanning electron microscopy of bone surfaces eroded in vivo showed trenches and pits of similar size as those made by OCs in culture. We conclude that the distinction between trench- and pit-forming OCs is relevant to the differences among OCs from different skeletal sites, different individuals, including gender, and results from differences in collagenolytic power. This indicates a biological relevance and highlights the importance of discriminating between pits and trenches when assessing resorption.

A reversal phase arrest uncoupling the bone formation and resorption contributes to the bone loss in glucocorticoid treated ovariectomised aged sheep

Large animals as sheep are often used as models for human osteoporosis. Our aim was therefore to determine how glucocorticoid treatment of ovariectomised sheep affects the cancellous bone, determining the cellular events within the bone remodelling process that contributes to their bone loss. Twenty female sheep were assigned for two groups; an untreated control group and an ovariectomised group treated with glucocorticoids (0.6 mg/kg/day, 5 times weekly) for 7 months. At 7 months the glucocorticoid-treated ovariectomised sheep showed a significant change in the bone microstructure revealed by a decreased trabecular bone volume and thickness compared to the control sheep. The treatment led to a temporary elevation of the bone resorption marker CTX (c-terminal collagen telopeptide), while the bone formation marker osteocalcin remained suppressed all 7 months. Histomorphometrically, the treated sheep had a complete absence of osteoid surfaces, and a 5-fold increase in the extent of eroded/reversal surfaces after 7 months. Most of these reversal surfaces were actually arrested reversal surfaces, defined as reversal surfaces without the presence of neighbouring osteoid surfaces or osteoclasts, which is classically observed next to active reversal surfaces. As in humans, these arrested reversal surfaces had compared to active reversal surfaces a reduced canopy coverage, a significantly decreased cell density, and a decreased immunoreactivity for the osteoblastic markers osterix, runx2 and smooth muscle actin in the mononuclear reversal cells colonising the surfaces. In conclusion, glucocorticoid treatment of ovariectomised sheep induced a significant bone loss, caused by an arrest of the reversal phase, resulting in an uncoupling of the bone formation and resorption during the reversal phase, as recently demonstrated in postmenopausal women with glucocorticoid-induced osteoporosis. This supports the relevance of the sheep model to the pathophysiology of glucocorticoid-induced osteoporosis in postmenopausal women, making it a relevant preclinical model for orthopaedic implant and biomaterial research.

Efficacy of a small cell-binding peptide coated hydroxyapatite substitute on bone formation and implant fixation in sheep.

Cylindrical critical size defects were created at the distal femoral condyles bilaterally of eight female adult sheep. Titanium implants with 2-mm concentric gaps were inserted and the gaps were filled with one of the four materials: allograft; a synthetic 15-amino acid cell-binding peptide coated hydroxyapatite (ABM/P-15); hydroxyapatite + βtricalciumphosphate+ Poly-Lactic-Acid (HA/βTCP-PDLLA); or ABM/P-15+HA/βTCP-PDLLA. After nine weeks, bone-implant blocks were harvested and sectioned for micro-CT scanning, push-out test, and histomorphometry. Significant bone formation and implant fixation could be observed in all four groups. Interestingly, the microarchitecture of the ABM/P-15 group was significantly different from the control group. Tissue volume fraction and thickness were significantly greater in the ABM/P-15 group than in the allograft group. Bone formation and bone ingrowth to porous titanium implant were not significantly different among the four groups. The ABM/P-15 group had similar shear mechanical properties on implant fixation as the allograft group. Adding HA/βTCP-PDLLA to ABM/P-15 did not significantly change these parameters. This study revealed that ABM/P-15 had significantly bone formation in concentric gap, and its enhancements on bone formation and implant fixation were at least as good as allograft. It is suggested that ABM/P-15 might be a good alternative biomaterial for bone implant fixation in this well-validated critical-size defect gap model in sheep. Nevertheless, future clinical researches should focus on prospective, randomized, controlled trials in order to fully elucidate whether ABM/P-15 could be a feasible candidate for bone substitute material in orthopedic practices.

Effects of substitute coated with hyaluronic acid or poly-lactic acid on implant fixation. Experimental study in ovariectomized and glucocorticoid treated sheep

Investigated in healthy animal models, hyaluronic acid (HyA) and poly‐D,L‐lactic acid (PDLLA) demonstrate osteoconductive properties when coated onto hydroxyapatite (HA) and β‐tricalcium phosphate (βTCP) scaffolds. In this study, we examined the efficacy of HA/βTCP granules coated with HyA or PDLLA on implant fixation when applied as graft materials in 2‐mm size defects created in the femur condyles of ovariectomized (OVX) glucocorticoid‐treated (GC) sheep. Titanium alloys were inserted into the femur condyles of OVX and GC‐treated sheep, and the concentric gaps were filled with either allograft obtained from a healthy donor sheep (control), pure HA/βTCP, HA/βTCP‐HyA or HA/βTCP‐PDLLA. After 12 weeks, the bone formation adjacent to the implant surface was evaluated by histology and histomorphometry, while the implant fixation was measured by a push‐out test. The investigation showed a bone formation in the HA/βTCP‐HyA and HA/βTCP‐PDLLA groups not significantly different from allograft (p  >  0.05), whereas the HA/βTCP group revealed a significantly reduced formation of bone compared with allograft (p  <  0.05). Bone–implant contact (BIC) and mechanical properties were similar comparing HA/βTCP‐HyA and HA/βTCP‐PDLLA with allograft (p  >  0.05). This study demonstrated that bone substitutes infiltrated with PDLLA and HyA possess osteoconductive properties comparable to allograft when tested in sheep with an OVX and GC‐induced bone loss. With no significant difference in implant fixation and bone formation, HyA and PDDLA are indeed considered valuable as new coating materials for composite ceramics when tested in a sheep model – even in bones of a compromised quality.

Intracortical Bone Mechanics Are Related to Pore Morphology and Remodeling in Human Bone

During aging and in osteoporosis, cortical bone becomes more porous, making it more fragile and susceptible to fractures. The aim of this study was to investigate the intracortical compression‐ induced strain energy distribution, and determine whether intracortical pores associated with high strain energy density (SED) in the surrounding bone matrix have a different morphology and distribution, as well as different remodeling characteristics than matrix with normal SED. Fibular diaphyseal specimens from 20 patients undergoing a jaw reconstruction (age range 41 to 75 years; 14 men and 6 women) were studied. Bone specimens were µCT‐scanned, plastic embedded, and sectioned for histology. Three‐dimensional microfinite element models of each specimen were tested in compression, and the SED of the bone immediately surrounding the intracortical pores was calculated within a plane of interest corresponding to the histological sections. The SED of a pore, relative to the distribution of the SED of all pores in each specimen, was used to classify pores as either a high or normal SED pore. Pores with high SED were larger, less circular, and were located closer to the endosteal surface of the cortex than normal SED pores (p < 0.001). Histological analysis of the remodeling events generating the pores revealed that the high SED pores compared with normal SED pores had 13.3‐fold higher odds of being an erosive (70%) or formative (7%) pore versus a quiescent pore (p < 0.001), 5.9‐fold higher odds of resulting from remodeling upon existing pores (type 2 pore) versus remodeling generating new pores (type 1 pore) (p < 0.001), and 3.2‐fold higher odds of being a coalescing type 2 pore versus a noncoalescing type 2 pore (p < 0.001). Overall, the study demonstrates a strong relationship between cortical bone mechanics and pore morphology, distribution, and remodeling characteristics in human fibular bone.

Understanding age-induced cortical porosity in women: Is a negative BMU balance in quiescent osteons a major contributor?

Cortical bone is remodeled by intracortical basic multicellular units (BMUs), whose end result can be observed as quiescent osteons in histological sections. These osteons offer a unique opportunity to investigate the BMU balance between the magnitude of bone resorption and subsequent bone formation at the BMU level. Our main objective was to investigate whether the latter parameters change between defined categories of osteons and with age, and to which extend these changes contribute to age-induced cortical porosity. Cortices of iliac bone specimens from 35 women (aged 16-78 years) with a higher porosity with age were investigated. A total of 3084 quiescent osteons reflecting 75% of the intracortical pores were histological examined. The osteons diameter, pore diameter, wall thickness, prevalence and contribution to the porosity were highly variable, but unchanged with age. Next, the osteons were categorized according to whether they reflected the remodeling of existing canals (type 2Q osteons) or the generation of new canals (type 1Q osteons). Type 2Q osteons versus type 1Q osteons: (i) had more frequently a pore diameter > 75 μm (7.4 vs. 1.3%; p < 0.001); (ii) had a larger mean pore diameter (40 ± 10 vs. 25 ± 4 μm; p < 0.001), osteon diameter (120 ± 21 vs. 94 ± 21 μm; p < 0.001) and wall thickness (40 ± 10 vs. 35 ± 9; p < 0.05); (iii) had a larger contribution to the cortical porosity (29 ± 18 vs. 8 ± 8%; p < 0.001); (iv) were more prevalent (44 ± 10 vs. 31 ± 11%; p < 0.001); and (v) were more prevalent with age. Collectively, this study demonstrates that quiescent osteons with age more frequently result from remodeling of existing canals, which in some cases had a more negative BMU balance. Still, the osteons showed no overall age-related change in their pore diameter i.e. BMU balance. In contrast to conventional wisdom, these data show that non-quiescent pores, not pores of quiescent osteons, were the main contributor to a higher cortical porosity.

The efficacy of poly-D,L-lactic acid- and hyaluronic acid-coated bone substitutes on implant fixation in sheep

Summary Background/Objective The present study investigated the efficacy of poly-d,l-lactic acid (PDLLA) and hyaluronic acid (HyA) on implant fixation when coated onto hydroxyapatite/beta-tri-calcium phosphate (HA/βTCP) granules. Methods The effect was assessed in a clinically relevant in vivo gap model in sheep. Thus, four titanium implants combined with either allograft (control), pure HA/βTCP, HyA infiltrated HA/βTCP, or PDLLA reinforced HA/βTCP granules were bilaterally inserted into the trabecular bone of the distal femurs in eight sheep. The insertion created a 2-mm peri-implant gap. After 12 weeks, histomorphometry and push-out test was used for quantification of newly formed bone in the gap, bone-implant contact, and implant fixation. Results The histomorphometric analysis revealed the presence of newly formed bone in all groups, though substitute groups showed fragments of nonabsorbed substitute material. A significant larger bone volume was found in the allograft group versus the HA/βTCP-PDLLA group (Zone 1), and in Zone 2 a statistically significantly larger bone volume was found in the allograft compared with the HA/βTCP group. The mechanical properties and the bone-implant contact revealed no statistically significant differences between the groups. Conclusion This study demonstrates that HA/βTCP granules coated with PDLLA and HyA have similar bone ingrowth and implant fixation as those with allograft, and with mechanical properties resembling those of allograft in advance, they may be considered as alternative substitute materials for bone formation in sheep.

Key intracortical remodeling events generating the increased cortical porosity during aging

Abstracts of the ECTS congress 2017s of the ECTS congress 2017 Published online: 11 May 2017

Erratum: Pit- and trench-forming osteoclasts: a distinction that matters.

[This corrects the article DOI: 10.1038/boneres.2015.32.].