High calcium bioglass enhances differentiation and survival of endothelial progenitor cells, inducing early vascularization in critical size bone defects

Early vascularization is a prerequisite for successful bone healing and endothelial progenitor cells (EPC), seeded on appropriate biomaterials, can improve vascularization. The type of biomaterial influences EPC function with bioglass evoking a vascularizing response. In this study the influence of a composite biomaterial based on polylactic acid (PLA) and either 20 or 40% bioglass, BG20 and BG40, respectively, on the differentiation and survival of EPCs in vitro was investigated. Subsequently, the effect of the composite material on early vascularization in a rat calvarial critical size defect model with or without EPCs was evaluated. Human EPCs were cultured with β-TCP, PLA, BG20 or BG40, and seeding efficacy, cell viability, cell morphology and apoptosis were analysed in vitro. BG40 released the most calcium, and improved endothelial differentiation and vitality best. This effect was mimicked by adding an equivalent amount of calcium to the medium and was diminished in the presence of the calcium chelator, EGTA. To analyze the effect of BG40 and EPCs in vivo, a 6-mm diameter critical size calvarial defect was created in rats (n = 12). Controls (n = 6) received BG40 and the treatment group (n = 6) received BG40 seeded with 5×105 rat EPCs. Vascularization after 1 week was significantly improved when EPCs were seeded onto BG40, compared to implanting BG40 alone. This indicates that Ca2+ release improves EPC differentiation and is useful for enhanced early vascularization in critical size bone defects

Cervical spine mechanical deficits in patients with shoulder impingement syndrome: A case-control study

The purpose of this case-control study was to investigate whether cervical mechanical deficits differ between patients with shoulder impingement syndrome (SIS) and control subjects. A total of 40 non-athletic males and females participated in this study. They were divided into two groups: group A, including 20 patients with SIS with a mean age of 36 years, and group B, including 20 control subjects with a mean age of 34 years. Each participant was assessed for pain and disability using shoulder pain and disability index (SPADI), active joint angular reproduction (AJAR) at 30o cervical flexion and extension using inclinometer, and craniovertebral angle (CVA) using photography. The results of the study showed that there was a statistically significant positive correlation between AJAR error at 30o cervical flexion and both shoulder pain and disability (r > 3, p = <0.05), and also a statically significant difference between groups at AJAR error in extension (t = 3.8, p = 0.000). In conclusion, cervical proprioception deficits showed a significant correlation with shoulder pain and disability in patients with SIS

Safety evaluation of a bioglass–polylactic acid composite scaffold seeded with progenitor cells in a rat skull critical-size bone defect

Treating large bone defects represents a major challenge in traumatic and orthopedic surgery. Bone tissue engineering provides a promising therapeutic option to improve the local bone healing response. In the present study tissue biocompatibility, systemic toxicity and tumorigenicity of a newly developed composite material consisting of polylactic acid (PLA) and 20% or 40% bioglass (BG20 and BG40), respectively, were analyzed. These materials were seeded with mesenchymal stem cells (MSC) and endothelial progenitor cells (EPC) and tested in a rat calvarial critical size defect model for 3 months and compared to a scaffold consisting only of PLA. Serum was analyzed for organ damage markers such as GOT and creatinine. Leukocyte count, temperature and free radical indicators were measured to determine the degree of systemic inflammation. Possible tumor occurrence was assessed macroscopically and histologically in slides of liver, kidney and spleen. Furthermore, the concentrations of serum malondialdehyde (MDA) and sodium oxide dismutase (SOD) were assessed as indicators of tumor progression. Qualitative tissue response towards the implants and new bone mass formation was histologically investigated. BG20 and BG40, with or without progenitor cells, did not cause organ damage, long-term systemic inflammatory reactions or tumor formation. BG20 and BG40 supported bone formation, which was further enhanced in the presence of EPCs and MSCs. This investigation reflects good biocompatibility of the biomaterials BG20 and BG40 and provides evidence that additionally seeding EPCs and MSCs onto the scaffold does not induce tumor formation

Surface structure and calcium release of the composite scaffold BG40.

<p>Disc shaped polymer/bioglass scaffold BG40, with a diameter of 5 mm and a thickness of 1 mm (A). A representative SEM image of BG40 is shown in (B). The composite demonstrates a relatively smooth surface with a thin fibrous texture and small pores (arrows). Scale bar indicates 6 µm. The release of ionic calcium to medium by BG40 (B4), BG20 (B2), PLA (P) and β-TCP (T) is shown in C. The most ionic calcium was released within the first two days. * = p<0.05 BG20, BG40 vs b-TCP, PLA; $ = p<0.05 BG40 vs BG20, n = 5.</p

Coincubation of EPCs with BG40 and BG20 leads to EPC elongation.

<p>Incubation with BG20 (B2) and BG40 (B4) lead to a significantly sustained increase in EPC length compared to EPCs incubated with β-TCP (T), PLA (P) or the medium control (C) during the whole observation period (A). The length of EPCs incubated with BG20 and BG40 increased significantly from day 2 to day 10 (A). Representative micrographs of EPCs incubated with either medium, PLA or BG40 demonstrated EPC elongation (B). * = p<0.05 BG20, BG40 vs control, β-TCP, PLA; $ = p<0.05 d6, d10 vs d2; # = β-TCP vs control, n = 5.</p

Gene expression of VEGF and vWF in EPC cultured on PLA, BG20 and BG40 over a period of 5 days.

<p>The values are given as fold change to the expression of the GAPDH gene which served as housekeeping gene (median value (25% quartile/75% quartile). Day 0 demonstrates the gene expression of the EPC before seeding on the biomaterials. Generally, gene epression of VEGF declined over the time whereas gene expression of vWF increased during the observation period. The experiment was performed with 4 independent EPC preparations. ↑ = increased gene expression; ↓ = decreased gene expression.</p

Calcium released by BG40 induces EPC elongation.

<p>EPCs were incubated in the presence of either 30/mL, 10 mM Ca²⁺ or BG40 conditioned medium+EGTA [3 mM]. Cell length was determined on day five (A) by means of phase contrast microscopy and subsequent histomorphometric evaluation. In (B) corresponding micrographs of EPCs are shown. The elongation of the EPCs was significantly lower in the presence of the calcium chelator EGTA [3 mM]. The uptake of DiL-ac-LDL demonstrated the endothelial phenotype and EPC vitality. The mean DiL-ac-LDL amount per cells was not significantly altered among the groups (control, 10 mM calcium, BG40, BG40+EGTA) (C). * = p<0.05 vs control; $ = p<0.05 vs BG40+EGTA, n = 5. Scale bars indicate 200 µm.</p

Serum biochemistry (part 1) after 3 months.

<p>Activities of transaminases (GOT/GPT), alkaline phosphatase (ALP) and creatinine is shown. Results are presented as median (25%-quartile/75%-quartile). EPC = endothelial progenitor cells, MSC = mesenchymal stem cells, dMSC = osteogenic predifferentiated MSC.</p