Combined application of BMP-2 and naturally occurring bioactive factor mixtures for the optimized therapy of segmental bone defects.

Critical bone defects are the result of traumatic, infection- or tumor-induced segmental bone loss and represent a therapeutic problem that has not been solved by current reconstructive or regenerative strategies yet. Scaffolds functionalized with naturally occurring bioactive factor mixtures show a promising chemotactic and angiogenic potential in vitro and therefore might stimulate bone regeneration in vivo. To assess this prospect, the study targets at heparin-modified mineralized collagen scaffolds functionalized with naturally occurring bioactive factor mixtures and/or rhBMP-2. These scaffolds were implanted into a 2-mm segmental femoral defect in mice and analyzed in respect to newly formed bone volume (BV) and bone mineral density (BMD) by micro-computed tomography scans after an observation period of 6 weeks. To rate the degree of defect healing, the number of vessels, and the activity of osteoclasts and osteoblasts were analyzed histologically. The sole application of bioactive factor mixtures is inferior to the use of the recombinant growth factor rhBMP-2 regarding BV and degree of defect healing. A higher rhBMP-2 concentration or the combination with bioactive factor mixtures does not lead to a further enhancement in defect healing. Possibly, a synergistic effect can be achieved by further concentration or a prolonged release of bioactive factor mixtures. STATEMENT OF SIGNIFICANCE: The successful therapy of extended bone defects is still a major challenge in clinical routine. In this study we investigated the bone regenerative potential of naturally occuring bioactive factor mixtures derived from platelet concentrates, adipose tissue and cell secretomes as a cheap and promising alternative to recombinant growth factors in a murine segmental bone defect model. The mixtures alone were not able to induce complete bridging of the bone defect, but in combination with bone morphogenetic protein 2 bone healing seemed to be more physiological. The results show that naturally occuring bioactive factor mixtures are a promising add-on in a clinical setting.

Chemotactic and Angiogenic Potential of Mineralized Collagen Scaffolds Functionalized with Naturally Occurring Bioactive Factor Mixtures to Stimulate Bone Regeneration.

To develop cost-effective and efficient bone substitutes for improved regeneration of bone defects, heparin-modified mineralized collagen scaffolds were functionalized with concentrated, naturally occurring bioactive factor mixtures derived from adipose tissue, platelet-rich plasma and conditioned medium from a hypoxia-treated human bone marrow-derived mesenchymal stem cell line. Besides the analysis of the release kinetics of functionalized scaffolds, the bioactivity of the released bioactive factors was tested with regard to chemotaxis and angiogenic tube formation. Additionally, functionalized scaffolds were seeded with human bone marrow-derived mesenchymal stromal cells (hBM-MSC) and their osteogenic and angiogenic potential was investigated. The release of bioactive factors from the scaffolds was highest within the first 3 days. Bioactivity of the released factors could be confirmed for all bioactive factor mixtures by successful chemoattraction of hBM-MSC in a transwell assay as well as by the formation of prevascular structures in a 2D co-culture system of hBM-MSC and human umbilical vein endothelial cells. The cells seeded directly onto the functionalized scaffolds were able to express osteogenic markers and form tubular networks. In conclusion, heparin-modified mineralized collagen scaffolds could be successfully functionalized with naturally occurring bioactive factor mixtures promoting cell migration and vascularization.

Cell spheroids are as effective as single cells suspensions in the treatment of critical-sized bone defects.

BACKGROUND: Due to their multilineage potential and high proliferation rate, mesenchymal stem cells (MSC) indicate a sufficient alternative in regenerative medicine. In comparison to the commonly used 2-dimensional culturing method, culturing cells as spheroids stimulates the cell-cell communication and mimics the in vivo milieu more accurately, resulting in an enhanced regenerative potential. To investigate the osteoregenerative potential of MSC spheroids in comparison to MSC suspensions, cell-loaded fibrin gels were implanted into murine critical-sized femoral bone defects. METHODS: After harvesting MSCs from 4 healthy human donors and preculturing and immobilizing them in fibrin gel, cells were implanted into 2 mm murine femoral defects and stabilized with an external fixator. Therefore, 26 14- to 15-week-old nu/nu NOD/SCID nude mice were randomized into 2 groups (MSC spheroids, MSC suspensions) and observed for 6 weeks. Subsequently, micro-computed tomography scans were performed to analyze regenerated bone volume and bone mineral density. Additionally, histological analysis, evaluating the number of osteoblasts, osteoclasts and vessels at the defect side, were performed. Statistical analyzation was performed by using the Student's t-test and, the Mann-Whitney test. The level of significance was set at p = 0.05. RESULTS: µCT-analysis revealed a significantly higher bone mineral density of the MSC spheroid group compared to the MSC suspension group. However, regenerated bone volume of the defect side was comparable between both groups. Furthermore, no significant differences in histological analysis between both groups could be shown. CONCLUSION: Our in vivo results reveal that the osteo-regenerative potential of MSC spheroids is similar to MSC suspensions.

IL-11 and soluble VCAM-1 are important components of Hypoxia Conditioned Media and crucial for Mesenchymal Stromal Cells attraction.

INTRODUCTION: Bone marrow stromal cells (BMSC) are highly attractive for tissue engineering due to their ability to differentiate into different cell types, to expand extensively in vitro and to release paracrine soluble factors with a high regenerative potential. They were observed to migrate towards the sites of injury in response to chemotactic signals in vivo. During the last years hypoxia has become a proven method to control proliferation, differentiation and multipotency of BMSC. Conditioned medium from hypoxia-treated BMSC (Hypoxia-conditioned Medium; HCM) has been shown to have various favorable properties on tissue regeneration - such as on cell recruitment, wound healing, angiogenesis and revascularization. Due to this regenerative potential many studies attempt to further characterize HCM and its main functional components. In this study we used HCM generated from umbilical cord mesenchymal stem cells (UC-MSC) instead of BMSC, because GMP-verified methods were used to isolate and cultivate the cells and ensure their constant quality. UC-MSC have a high regenerative potential and are still immunologically naive and therefore highly unlikely to cause an immune reaction. In our article we took the first steps to closer investigate the role of umbilical cord MSC-derived HCM components, namely stromal cell-derived factor 1 (SDF-1α), interleukin 11 (IL-11) and soluble vascular cell adhesion molecule 1 (sVCAM-1). RESULTS: Our results show previously unknown roles of IL-11 and sVCAM-1 in the attraction of BMSC. The synergistic effect of the investigated protein mixture consisting of IL-11, sVCAM-1 and SDF-1α as well as those recombinant proteins alone revealed a significantly higher chemoattractive capacity towards human BMSC compared to normoxic control medium. Both, the protein mixtures and proteins alone as well as UC-HCM showed an angiogenic effect by promoting the formation of significantly longer tubule structures and higher amounts of junctions and tubules compared to normoxic control medium. CONCLUSIONS: By showing the prominent upregulation of IL-11, sVCAM-1 and SDF-1α under hypoxic conditions compared to normoxic control and revealing their crucial role in migration of human BMSC we took a further step forward in characterization of the chemoattractive components of HCM.

Characterization of Naturally Occurring Bioactive Factor Mixtures for Bone Regeneration.

In this study, the bone-regenerative potential of bioactive factors derived from adipose tissue, platelet-rich plasma (PRP) and conditioned medium from hypoxia-treated human telomerase immortalized bone-marrow-derived mesenchymal stem cells (hTERT-MSC) was investigated in vitro with the aim to develop cost-effective and efficient bone substitutes for optimized regeneration of bone defects. Adipose tissue was harvested from human donors undergoing reconstructive surgery, and adipose tissue extract (ATE) was prepared. Platelet lysates (PL) were produced by repeated freeze-thaw cycles of PRP, and hypoxia-conditioned medium (HCM) was obtained by culturing human telomerase immortalized bone-marrow-derived mesenchymal stromal cells for 5 days with 1% O2. Besides analysis by cytokine and angiogenesis arrays, ELISA was performed. Angiogenic potential was investigated in cocultures of bone-marrow-derived (BM)-MSC and human umbilical vein endothelial cells. Multiple angiogenic proteins and cytokines were detected in all growth factor mixtures. HCM and ATE contained high amounts of angiogenin and CCL2/MCP-1, whereas PL contained high amounts of IGFBP-1. Culturing cells with HCM and ATE significantly increased specific ALP activity of BM-MSC as well as tubule length and junctions of endothelial networks, indicating osteogenic and angiogenic stimulation. To achieve a synergism between chemoattractive potential and osteogenic and angiogenic differentiation capacity, a combination of different growth factors appears promising for potential clinical applications.

Strontium enhances BMP-2 mediated bone regeneration in a femoral murine bone defect model.

The application of strontium is one option for the clinical treatment of osteoporosis-a disease characterized by reduced bone density and quality-in order to reduce the risk of vertebral and nonvertebral fractures. Unlike other drugs used in osteoporosis therapy, strontium shows a dual effect on bone metabolism by attenuating cellular resorption and simultaneously enhancing new bone tissue formation. Current concerns regarding the systemic application of highly dosed strontium ranelate led to the development of strontium-modified scaffolds based on mineralized collagen (MCM) capable to release biologically active Sr2+ ions directly at the fracture site. In this study, we investigated the regenerative potential of these scaffolds. For in vitro investigations, human mesenchymal stromal cells were cultivated on the scaffolds for 21 days (w/ and w/o osteogenic supplements). Biochemical analysis revealed a significant promoting effect on proliferation rate and osteogenic differentiation on strontium-modified scaffolds. In vivo, scaffolds were implanted in a murine segmental bone defect model-partly additionally functionalized with the osteogenic growth factor bone morphogenetic protein 2 (BMP-2). After 6 weeks, bridging calluses were obtained in BMP-2 functionalized scaffolds; the quality of the newly formed bone tissue by means of morphological scores was clearly enhanced in strontium-modified scaffolds. Histological analysis revealed increased numbers of osteoblasts and blood vessels, decreased numbers of osteoclasts, and significantly enhanced mechanical properties. These results indicate that the combined release of Sr2+ ions and BMP-2 from the biomimetic scaffolds is a promising strategy to enhance bone regeneration, especially in patients suffering from osteoporosis. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:174-182, 2020.

The Secretome of Hypoxia Conditioned hMSC Loaded in a Central Depot Induces Chemotaxis and Angiogenesis in a Biomimetic Mineralized Collagen Bone Replacement Material.

The development of biomaterials with intrinsic potential to stimulate endogenous tissue regeneration at the site of injury is a main demand on future implants in regenerative medicine. For critical-sized bone defects, an in situ tissue engineering concept is devised based on biomimetic mineralized collagen scaffolds. These scaffolds are functionalized with a central depot loaded with a signaling factor cocktail, obtained from secretome of hypoxia-conditioned human mesenchymal stem cells (MSC). Therefore, hypoxia-conditioned medium (HCM)-production is standardized and adapted to achieve high signaling factor-yields; a concentration protocol based on dialysis and freeze-drying is established to enable the integration of sufficient and defined amounts into the depot. In humid milieu-as after implantation-signaling factors are released by forming a chemotactic gradient, inducing a directed migration of human bone marrow stroma cells (hBMSC) into the scaffold. Angiogenic potential, determined by coculturing human umbilical vein endothelial cells (HUVEC) with osteogenically induced hBMSC shows prevascular structures, which sprout throughout the interconnected pores in a HCM-concentration-dependent manner. Retarded release by alginate-based (1 vol%) depots, significantly improves sprouting-depth and morphology of tubular structures. With the intrinsic potential to supply attracted cells with oxygen and nutrients, this bioactive material system has great potential for clinical translation.

Investigation of strontium transport and strontium quantification in cortical rat bone by time-of-flight secondary ion mass spectrometry.

Next-generation bone implants will be functionalized with drugs for stimulating bone growth. Modelling of drug release by such functionalized biomaterials and drug dispersion into bone can be used as predicting tool for biomaterials testing in future. Therefore, the determination of experimental parameters to describe and simulate drug release in bone is essential. Here, we focus on Sr2+ transport and quantification in cortical rat bone. Sr2+ dose-dependently stimulates bone-building osteoblasts and inhibits bone-resorbing osteoclasts. It should be preferentially applied in the case of bone fracture in the context of osteoporotic bone status. Transport properties of cortical rat bone were investigated by dipping experiments of bone sections in aqueous Sr2+ solution followed by time-of-flight secondary ion mass spectrometry (ToF-SIMS) depth profiling. Data evaluation was carried out by fitting a suitable mathematical diffusion equation to the experimental data. An average diffusion coefficient of D = (1.68 ± 0.57) · 10-13 cm2 s-1 for healthy cortical bone was obtained. This value differed only slightly from the value of D = (4.30 ± 1.43) · 10-13 cm2 s-1 for osteoporotic cortical bone. Transmission electron microscopy investigations revealed a comparable nano- and ultrastructure for both types of bone status. Additionally, Sr2+-enriched mineralized collagen standards were prepared for ToF-SIMS quantification of Sr2+ content. The obtained calibration curve was used for Sr2+ quantification in cortical and trabecular bone in real bone sections. The results allow important insights regarding the Sr2+ transport properties in healthy and osteoporotic bone and can ultimately be used to perform a simulation of drug release and mobility in bone.

3D Plotted Biphasic Bone Scaffolds for Growth Factor Delivery: Biological Characterization In Vitro and In Vivo.

Bioprinting enables the integration of biological components into scaffolds during fabrication that has the advantage of high loading efficiency and better control of release and/or spatial positioning. In this study, a biphasic scaffold fabricated by extrusion-based 3D multichannel plotting of a calcium phosphate cement (CPC) paste and an alginate/gellan gum (AlgGG) hydrogel paste laden with the angiogenic factor VEGF (vascular endothelial growth factor) is investigated with regard to biological response in vitro and in vivo. Rat mesenchymal stromal cells are able to adhere and grow on both CPC and AlgGG strands, and differentiate toward osteoblasts. A sustained VEGF release is observed, which is able to stimulate endothelial cell proliferation as well as angiogenesis in vitro that indicates maintenance of its biological activity. After implantation into a segmental bone defect in the femur diaphysis of rats, a clear reduction of the defect size by newly formed bone tissue occurs from the distal and proximal ends of the host bone within 12 weeks. The CPC component shows excellent osteoconductivity whereas the local VEGF release from the AlgGG hydrogel gives rise to an enhanced vascularization of the defect region. This work contributes to the development of novel therapeutic concepts for improved bone regeneration which are based on 3D bioprinting.

Influence of Regioselectively Sulfated Cellulose on in Vitro Vascularization of Biomimetic Bone Matrices.

Vascularization is essential for the regeneration of bone tissue within composite material. We measured the effect of regioselectively modified cellulose/hemicellulose as an additive for porous scaffolds of collagen/hydroxyapatite nanocomposite on the tubule formation of human vascular endothelial cells. Using a coculture of endothelial cells and fibroblasts, endothelial cells formed a network of tubules within an incubation time of 14 to 24 days. A cellulose sulfate with irregular sulfation pattern along the polysaccharide backbone (13-TACS-01) led to an additional increase in vascular endothelial growth factor (VEGF)-induced tubule formation, as observed in an in vitro angiogenesis assays. In contrast with structurally different heparin, these cellulose sulfates have no apparent affinity to VEGF. Their impact on endothelial function may possibly be due to interactions with cell surface receptors/soluble factors not yet defined.

Strontium-modification of porous scaffolds from mineralized collagen for potential use in bone defect therapy.

The present study describes the development and characterization of strontium(II)-modified biomimetic scaffolds based on mineralized collagen type I as potential biomaterial for the local treatment of defects in systemically impaired (e.g. osteoporotic) bone. In contrast to already described collagen/hydroxyapatite nanocomposites calcium was substituted with strontium to the extent of 25, 50, 75 and 100mol% by substituting the CaCl2-stock solution (0.1M) with SrCl2 (0.1M) during the scaffold synthesis. Simultaneous fibrillation and mineralization of collagen led to the formation of collagen-mineral nanocomposites with mineral phases shifting from nanocrystalline hydroxyapatite (Sr0) over poorly crystalline Sr-rich phases towards a mixed mineral phase (Sr100), consisting of an amorphous strontium phosphate (identified as Collin's salt, Sr6H3(PO4)5∗2 H2O, CS) and highly crystalline strontium hydroxyapatite (Sr5(PO4)3OH, SrHA). The formed mineral phases were characterized by transmission electron microscopy (TEM) and RAMAN spectroscopy. All collagen/mineral nanocomposites with graded strontium content were processed to scaffolds exhibiting an interconnected porosity suitable for homogenous cell seeding in vitro. Strontium ions (Sr2+) were released in a sustained manner from the modified scaffolds, with a clear correlation between the released Sr2+ concentration and the degree of Sr-substitution. The accumulated specific Sr2+ release over the course of 28days reached 141.2µg (~27µgmg-1) from Sr50 and 266.1µg (~35µgmg-1) from Sr100, respectively. Under cell culture conditions this led to maximum Sr2+ concentrations of 0.41mM (Sr50) and 0.73mM (Sr100) measured on day 1, which declined to 0.08mM and 0.16mM, respectively, at day 28. Since Sr2+ concentrations in this range are known to have an osteo-anabolic effect, these scaffolds are promising biomaterials for the clinical treatment of defects in systemically impaired bone.

* Central Growth Factor Loaded Depots in Bone Tissue Engineering Scaffolds for Enhanced Cell Attraction.

Tissue engineering, the application of stem and progenitor cells in combination with an engineered extracellular matrix, is a promising strategy for bone regeneration. However, its success is limited by the lack of vascularization after implantation. The concept of in situ tissue engineering envisages the recruitment of cells necessary for tissue regeneration from the host environment foregoing ex vivo cell seeding of the scaffold. In this study, we developed a novel scaffold system for enhanced cell attraction, which is based on biomimetic mineralized collagen scaffolds equipped with a central biopolymer depot loaded with chemotactic agents. In humid milieu, as after implantation, the signaling factors are expected to slowly diffuse out of the central depot forming a gradient that stimulates directed cell migration toward the scaffold center. Heparin, hyaluronic acid, and alginate have been shown to be capable of depot formation. By using vascular endothelial growth factor (VEGF) as model factor, it was demonstrated that the release kinetics can be adjusted by varying the depot composition. While alginate and hyaluronic acid are able to reduce the initial burst and prolong the release of VEGF, the addition of heparin led to a much stronger retention that resulted in an almost linear release over 28 days. The biological activity of released VEGF was proven for all variants using an endothelial cell proliferation assay. Furthermore, migration experiments with endothelial cells revealed a relationship between the degree of VEGF retention and migration distance: cells invaded deepest in scaffolds containing a heparin-based depot indicating that the formation of a steep gradient is crucial for cell attraction. In conclusion, this novel in situ tissue engineering approach, specifically designed to recruit and accommodate endogenous cells upon implantation, appeared highly promising to stimulate cell invasion, which in turn would promote vascularization and finally new bone formation.

Cu2+, Co2+ and Cr3+ doping of a calcium phosphate cement influences materials properties and response of human mesenchymal stromal cells.

The application of biologically active metal ions to stimulate cellular reactions is a promising strategy to accelerate bone defect healing. Brushite-forming calcium phosphate cements were modified with low doses of Cu2+, Co2+ and Cr3+. The modified cements released the metal ions in vitro in concentrations which were shown to be non-toxic for cells. The release kinetics correlated with the solubility of the respective metal phosphates: 17-45 wt.-% of Co2+ and Cu2+, but <1 wt.-% of Cr3+ were released within 28days. Moreover, metal ion doping led to alterations in the exchange of calcium and phosphate ions with cell culture medium. In case of cements modified with 50mmol Cr3+/mol ß-tricalcium phosphate (ß-TCP), XRD and SEM analyses revealed a significant amount of monetite and a changed morphology of the cement matrix. Cell culture experiments with human mesenchymal stromal cells indicated that the observed cell response is not only influenced by the released metal ions but also by changed cement properties. A positive effect of modifications with 50mmol Cr3+ or 10mmol Cu2+ per mol ß-TCP on cell behaviour was observed in indirect and direct culture. Modification with Co2+ resulted in a clear suppression of cell proliferation and osteogenic differentiation. In conclusion, metal ion doping of the cement influences cellular activities in addition to the effect of released metal ions by changing properties of the ceramic matrix.

A modular segmented-flow platform for 3D cell cultivation.

In vitro 3D cell cultivation is promised to equate tissue in vivo more realistically than 2D cell cultivation corresponding to cell-cell and cell-matrix interactions. Therefore, a scalable 3D cultivation platform was developed. This platform, called pipe-based bioreactors (pbb), is based on the segmented-flow technology: aqueous droplets are embedded in a water-immiscible carrier fluid. The droplet volumes range from 60 nL to 20 µL and are used as bioreactors lined up in a tubing like pearls on a string. The modular automated platform basically consists of several modules like a fluid management for a high throughput droplet generation for self-assembly or scaffold-based 3D cell cultivation, a storage module for incubation and storage, and an analysis module for monitoring cell aggregation and proliferation basing on microscopy or photometry. In this report, the self-assembly of murine embryonic stem cells (mESCs) to uniformly sized embryoid bodies (EBs), the cell proliferation, the cell viability as well as the influence on the cell differentiation to cardiomyocytes are described. The integration of a dosage module for medium exchange or agent addition will enable pbb as long-term 3D cell cultivation system for studying stem cell differentiation, e.g. cardiac myogenesis or for diagnostic and therapeutic testing in personalized medicine.

Mobile applications for diabetics: a systematic review and expert-based usability evaluation considering the special requirements of diabetes patients age 50 years or older.

BACKGROUND: A multitude of mhealth (mobile health) apps have been developed in recent years to support effective self-management of patients with diabetes mellitus type 1 or 2. OBJECTIVE: We carried out a systematic review of all currently available diabetes apps for the operating systems iOS and Android. We considered the number of newly released diabetes apps, range of functions, target user groups, languages, acquisition costs, user ratings, available interfaces, and the connection between acquisition costs and user ratings. Additionally, we examined whether the available applications serve the special needs of diabetes patients aged 50 or older by performing an expert-based usability evaluation. METHODS: We identified relevant keywords, comparative categories, and their specifications. Subsequently, we performed the app review based on the information given in the Google Play Store, the Apple App Store, and the apps themselves. In addition, we carried out an expert-based usability evaluation based on a representative 10% sample of diabetes apps. RESULTS: In total, we analyzed 656 apps finding that 355 (54.1%) offered just one function and 348 (53.0%) provided a documentation function. The dominating app language was English (85.4%, 560/656), patients represented the main user group (96.0%, 630/656), and the analysis of the costs revealed a trend toward free apps (53.7%, 352/656). The median price of paid apps was €1.90. The average user rating was 3.6 stars (maximum 5). Our analyses indicated no clear differences in the user rating between free and paid apps. Only 30 (4.6%) of the 656 available diabetes apps offered an interface to a measurement device. We evaluated 66 apps within the usability evaluation. On average, apps were rated best regarding the criterion "comprehensibility" (4.0 out of 5.0), while showing a lack of "fault tolerance" (2.8 out of 5.0). Of the 66 apps, 48 (72.7%) offered the ability to read the screen content aloud. The number of functions was significantly negative correlated with usability. The presence of documentation and analysis functions reduced the usability score significantly by 0.36 and 0.21 points. CONCLUSIONS: A vast number of diabetes apps already exist, but the majority offer similar functionalities and combine only one to two functions in one app. Patients and physicians alike should be involved in the app development process to a greater extent. We expect that the data transmission of health parameters to physicians will gain more importance in future applications. The usability of diabetes apps for patients aged 50 or older was moderate to good. But this result applied mainly to apps offering a small range of functions. Multifunctional apps performed considerably worse in terms of usability. Moreover, the presence of a documentation or analysis function resulted in significantly lower usability scores. The operability of accessibility features for diabetes apps was quite limited, except for the feature "screen reader".