Early pH Changes in Musculoskeletal Tissues upon Injury-Aerobic Catabolic Pathway Activity Linked to Inter-Individual Differences in Local pH.

Local pH is stated to acidify after bone fracture. However, the time course and degree of acidification remain unknown. Whether the acidification pattern within a fracture hematoma is applicable to adjacent muscle hematoma or is exclusive to this regenerative tissue has not been studied to date. Thus, in this study, we aimed to unravel the extent and pattern of acidification in vivo during the early phase post musculoskeletal injury. Local pH changes after fracture and muscle trauma were measured simultaneously in two pre-clinical animal models (sheep/rats) immediately after and up to 48 h post injury. The rat fracture hematoma was further analyzed histologically and metabolomically. In vivo pH measurements in bone and muscle hematoma revealed a local acidification in both animal models, yielding mean pH values in rats of 6.69 and 6.89, with pronounced intra- and inter-individual differences. The metabolomic analysis of the hematomas indicated a link between reduction in tricarboxylic acid cycle activity and pH, thus, metabolic activity within the injured tissues could be causative for the different pH values. The significant acidification within the early musculoskeletal hematoma could enable the employment of the pH for novel, sought-after treatments that allow for spatially and temporally controlled drug release.

Hydrogels with tunable stress relaxation regulate stem cell fate and activity.

Natural extracellular matrices (ECMs) are viscoelastic and exhibit stress relaxation. However, hydrogels used as synthetic ECMs for three-dimensional (3D) culture are typically elastic. Here, we report a materials approach to tune the rate of stress relaxation of hydrogels for 3D culture, independently of the hydrogel's initial elastic modulus, degradation, and cell-adhesion-ligand density. We find that cell spreading, proliferation, and osteogenic differentiation of mesenchymal stem cells (MSCs) are all enhanced in cells cultured in gels with faster relaxation. Strikingly, MSCs form a mineralized, collagen-1-rich matrix similar to bone in rapidly relaxing hydrogels with an initial elastic modulus of 17 kPa. We also show that the effects of stress relaxation are mediated by adhesion-ligand binding, actomyosin contractility and mechanical clustering of adhesion ligands. Our findings highlight stress relaxation as a key characteristic of cell-ECM interactions and as an important design parameter of biomaterials for cell culture.

Matrix elasticity of void-forming hydrogels controls transplanted-stem-cell-mediated bone formation.

The effectiveness of stem cell therapies has been hampered by cell death and limited control over fate. These problems can be partially circumvented by using macroporous biomaterials that improve the survival of transplanted stem cells and provide molecular cues to direct cell phenotype. Stem cell behaviour can also be controlled in vitro by manipulating the elasticity of both porous and non-porous materials, yet translation to therapeutic processes in vivo remains elusive. Here, by developing injectable, void-forming hydrogels that decouple pore formation from elasticity, we show that mesenchymal stem cell (MSC) osteogenesis in vitro, and cell deployment in vitro and in vivo, can be controlled by modifying, respectively, the hydrogel's elastic modulus or its chemistry. When the hydrogels were used to transplant MSCs, the hydrogel's elasticity regulated bone regeneration, with optimal bone formation at 60 kPa. Our findings show that biophysical cues can be harnessed to direct therapeutic stem cell behaviours in situ.

The 24th annual meeting of the European Tissue Repair Society (ETRS) in Edinburgh, Scotland.

From the 10th to 12th of September 2014, in the midst of the Scottish Independence debate, the European Tissue Repair Society descended on Edinburgh for their 24th Annual Meeting. In the beautiful and historic setting of the Royal College of Surgeons of Scotland, Professors David Thomas (Chair), Phil Stephens, Chris Lloyd, and their teams from Cardiff hosted an educational and inspiring program.

Role of extracellular matrix structural components and tissue mechanics in the development of postoperative pancreatic fistula.

Radical resection remains the only curative treatment option in pancreatic cancer. Postoperative pancreatic fistulas (POPF) occur in up to 30% of patients leading to prolonged hospital-stay, increased cost of care and morbidity and mortality. Mechanical properties of the pancreas are associated with POPF. The aim of this study is to analyze the role of extracellular matrix (ECM) and tissue mechanics in the risk of POPF. Biopsies of 41 patients receiving a partial pancreas-resection are analyzed. Clinical data, ECM components and mechanical properties are correlated with POPF. Preoperative cholestasis is correlated with reduced risk of POPF, which comes along with a dilatation of the pancreatic duct and significantly higher content of collagen I. Patients developing POPF exhibited a degenerated tissue integrity, with significantly lower content of fibronectin and a trend for lower collagen I, III, IV and hyaluronic acid. This correlated with a soft tactile sensation of the surgeon during the intervention. However, this was not reflected with tissue mechanics evaluated by ex vivo uniaxial compression testing, where a significantly higher elastic modulus and no effect on the stress relaxation time were found. In conclusion, patients with cholestasis seem to have a lower risk for POPF, and an increase in collagen I. A degenerated matrix with lower content of structural ECM components correlates with increased risk of POPF. However, ex vivo uniaxial compression testing failed to clearly explain the link of ECM properties and POPF.

Slow cooling cryopreservation of cell-microcarrier constructs.

Ideally, tissue engineered constructs should be readily available to meet the need for fast intervention in complex bone defects. To circumvent the long culture period of these constructs before implantation, we investigated the possibility of cryopreserving cell-loaded constructs. Goat bone marrow-derived mesenchymal stem cells (BMSC) and mouse osteoblast-like cells from the MC3T3-E1 cell line were cultured on gelatin CultiSpher-S(R) microcarriers. These constructs were cryopreserved using the slow cooling technique, i.e. cooling to -80 degrees C at a rate of -1.5 degrees C/min, and were then stored in liquid nitrogen for 1 week. Four different cryomedia were tested, i.e. 90 vol% serum with 10 vol% dimethylsulphoxide (Me(2)SO) with or without ascorbic acid (AA) and 90 vol% serum supplemented with 5 vol% Me(2)SO and 5 vol% hydroxyethyl starch or 5 vol% sucrose (60 mM). Cell viability on the constructs was assessed with fluorescent live/dead staining and the colorimetric MTS assay. Cell viability was compared before freezing and at fixed time points after thawing. Immediately after thawing, the viability percentages in all groups were significantly lower than before cryopreservation (p = 0.0369). No significant differences were observed between the viability percentages on the cell constructs cryopreserved in the different media; however, there was a general tendency for higher cell survival and faster recolonization of constructs cryopreserved in Me(2)SO with or without AA than of the constructs cryopreserved in the other media. For constructs cryopreserved in 10 vol% Me(2)SO with or without AA, the recolonization period was 3 days for the BMSC constructs and 3.6 and 3.8 days, respectively, for the MC3T3-E1 constructs.

Oxidized alginate beads for tunable release of osteogenically potent mesenchymal stromal cells.

Bone defect repair can benefit from local delivery of mesenchymal stromal cells (MSCs). However, local harsh environmental conditions after injury may necessitate a cell therapy strategy that shields MSCs initially and releases them locally over time. This may be possible by using biomaterials that exhibit stimuli-responsive degradability, such as oxidized alginate hydrogels that undergo hydrolytic degradation. However, it remains unknown whether varying encapsulation periods compromise MSC osteogenic differentiation capacity after release. To address this, we cultured MSCs in 3D alginate beads with tunable degradability before characterizing the function of released cells. Alginates were oxidized to different degrees (2%, 3%, and 4%) to achieve distinct rates of degradation (days to weeks), then functionalized with RGD peptides to enable cell adhesion, and modified additionally with 6-aminofluorescin to enable fluorescence-based detection. Bead morphology, degradation kinetics, cell morphology, and cell release kinetics were monitored over time. Cells that were released from the beads were stimulated to differentiate into the osteogenic lineage. Our results indicate that MSCs released from all bead groups retained a strong ability to deposit mineralized matrix under osteogenic differentiation conditions. These findings provide the basis for designing and implementing biomaterial-based strategies for the in-situ temporal delivery of potent MSCs at bone defect sites.

Collagen Fibrils Mechanically Contribute to Tissue Contraction in an In Vitro Wound Healing Scenario.

Wound contraction is an ancient survival mechanism of vertebrates that results from tensile forces supporting wound closure. So far, tissue tension was attributed to cellular forces produced by tissue-resident (myo-)fibroblasts alone. However, difficulties in explaining pathological deviations from a successful healing path motivate the exploration of additional modulatory factors. Here, it is shown in a biomaterial-based in vitro wound healing model that the storage of tensile forces in the extracellular matrix has a significant, so-far neglected contribution to macroscopic tissue tension. In situ monitoring of tissue forces together with second harmonic imaging reveal that the appearance of collagen fibrils correlates with tissue contraction, indicating a mechanical contribution of tensioned collagen fibrils in the contraction process. As the re-establishment of tissue tension is key to successful wound healing, the findings are expected to advance the understanding of tissue healing but also underlying principles of misregulation and impaired functionality in scars and tissue contractures.

Dosage and composition of bioactive glasses differentially regulate angiogenic and osteogenic response of human MSCs.

Vascularization of the fracture site and cell-mediated deposition of the mineralized matrix are crucial determinants for successful bone regeneration after injury. Ceramic biomaterials such as bioactive glasses (BAGs) that release bioactive ions have shown promising results in bone defect regeneration. However, it remains unclear how the dosage and composition of bioactive ions influence the angiogenic and osteogenic behavior of primary human mesenchymal stromal cells (MSCs). Here, we show that exposure to ionic dissolution products from 1393 and 45S5 BAGs can evoke distinct angiogenic and osteogenic responses from primary MSCs in a dose- and composition-dependent manner. Significantly higher concentrations of the pro-angiogenic factors VEGF, HGF, PIGF, angiopoietin, and angiogenin were detected in conditioned media (CM) from MSCs exposed to 45S5, but not 1393, BAGs. Application of this CM to human umbilical vein endothelial cells (HUVECs) resulted in robust 2D tube formation in vitro. Osteogenic differentiation of MSCs was assessed by gene expression analysis and mineralization assays. Low concentrations (0.1% w/v) of 1393 BAGs significantly enhanced the gene expression of RUNX2 and ALP and induced an earlier onset of matrix mineralization compared to all other groups. We further tested whether simultaneous exposure to both BAGs would improve both angiogenic secretion and osteogenic differentiation of MSCs, and did not find evidence to support this hypothesis. Our results provide evidence of BAG composition-dependent enhancement of primary human MSCs' regenerative function, besides also underlining the importance of an in vitro evaluation of the dose-response relationship to translate BAG based approaches into safe and effective clinical therapies. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2827-2837, 2018., 2018.

Comparison of the effects of 45S5 and 1393 bioactive glass microparticles on hMSC behavior.

Bioactive glasses (BAGs) are highly interesting materials for bone regeneration applications in orthopedic and dental defects. It is quite well known that ionic release from BAGs influences cell behavior and function. Mindful of the clinical scenario, we hypothesized that local cell populations might additionally physically interact with the implanted BAG particles and respond differently than to just the ionic stimuli. We therefore studied the biological effect of two BAG types (45S5 and 1393) applied to human mesenchymal stromal cells (hMSCs) in three distinct presentation modes: (a) direct contact; and to dissolution products in (b) 2D, and (c) 3D culture. We furthermore investigated how the dose-dependence of these BAG particles, in concentrations ranging from 0.1 to 2.5 w/v %, influenced hMSC metabolic activity, proliferation, and cell spreading. These cellular functions were significantly hampered when hMSCs were exposed to high concentrations of either glasses, but the effects were more pronounced in the 45S5 groups and when the cells were in direct contact with the BAGs. Furthermore the biological effect of 1393 BAG outperformed that of 45S5 BAG in all tested presentation modes. These outcomes highlight the importance of investigating cell-BAG interactions in experimental set-ups that recapitulate host cell interactions with BAG particles. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2772-2782, 2017.

Substrate Stress-Relaxation Regulates Scaffold Remodeling and Bone Formation In Vivo.

The rate of stress relaxation of adhesion substrates potently regulates cell fate and function in vitro, and in this study the authors test whether it can regulate bone formation in vivo by implanting alginate gels with differing rates of stress-relaxation carrying human mesenchymal stem cells into rat calvarial defects. After three months, the rats that received fast-relaxing hydrogels (t1/2 ≈ 50 s) show significantly more new bone growth than those that received slow-relaxing, stiffness-matched hydrogels. Strikingly, substantial bone regeneration results from rapidly relaxing hydrogels even in the absence of transplanted cells. Histological analysis reveals that the new bone formed with rapidly relaxing hydrogels is mature and accompanied by extensive matrix remodeling and hydrogel disappearance. This tissue invasion is found to be prominent after just two weeks and the ability of stress relaxation to modulate cell invasion is confirmed with in vitro analysis. These results suggest that substrate stress relaxation can mediate scaffold remodeling and thus tissue formation, giving tissue engineers a new parameter for optimizing bone regeneration.

Bone regeneration via novel macroporous CPC scaffolds in critical-sized cranial defects in rats.

OBJECTIVES: Calcium phosphate cement (CPC) is promising for dental and craniofacial applications due to its ability to be injected or filled into complex-shaped bone defects and molded for esthetics, and its resorbability and replacement by new bone. The objective of this study was to investigate bone regeneration via novel macroporous CPC containing absorbable fibers, hydrogel microbeads and growth factors in critical-sized cranial defects in rats. METHODS: Mannitol porogen and alginate hydrogel microbeads were incorporated into CPC. Absorbable fibers were used to provide mechanical reinforcement to CPC scaffolds. Six CPC groups were tested in rats: (1) control CPC without macropores and microbeads; (2) macroporous CPC+large fiber; (3) macroporous CPC+large fiber+nanofiber; (4) same as (3), but with rhBMP2 in CPC matrix; (5) same as (3), but with rhBMP2 in CPC matrix+rhTGF-ß1 in microbeads; (6) same as (3), but with rhBMP2 in CPC matrix+VEGF in microbeads. Rats were sacrificed at 4 and 24 weeks for histological and micro-CT analyses. RESULTS: The macroporous CPC scaffolds containing porogen, absorbable fibers and hydrogel microbeads had mechanical properties similar to cancellous bone. At 4 weeks, the new bone area fraction (mean±sd; n=5) in CPC control group was the lowest at (14.8±3.3)%, and that of group 6 (rhBMP2+VEGF) was (31.0±13.8)% (p<0.05). At 24 weeks, group 4 (rhBMP2) had the most new bone of (38.8±15.6)%, higher than (12.7±5.3)% of CPC control (p<0.05). Micro-CT revealed nearly complete bridging of the critical-sized defects with new bone for several macroporous CPC groups, compared to much less new bone formation for CPC control. SIGNIFICANCE: Macroporous CPC scaffolds containing porogen, fibers and microbeads with growth factors were investigated in rat cranial defects for the first time. Macroporous CPCs had new bone up to 2-fold that of traditional CPC control at 4 weeks, and 3-fold that of traditional CPC at 24 weeks, and hence may be useful for dental, craniofacial and orthopedic applications.

Biocompatibility properties of surface-modified poly(dimethylsiloxane) for urinary applications.

An electronic sensor system for urinary bladder pressure monitoring requires an imbedding into a biocompatible, flexible, and liquid-impermeable material. Poly(dimethylsiloxane) (PDMS) was selected in the present set-up as packaging material because it fulfills the abovementioned requirements. However, the surface of PDMS is hydrophobic and causes undesired interactions with salts, proteins, and cells present in urine. To reduce possible interactions of urine salts in the urinary bladder, monomers, [2-(methacryloyloxy)ethyl]-dimethyl-3-sulfopropyl-ammonium hydroxide (sulfobetaine) and 2-acrylamido-2-methylpropyl sulfonic acid, were grafted onto the surface through oxygen plasma treatment. A reduction in salt deposition between the pure PDMS and the modified PDMS was observed both in vitro (artificial urine flow over the surface) and in vivo (implants into the urinary bladder of experimental pigs). Additionally, a 10-fold reduction in salt deposition was observed in vitro due to grafting of the monomers onto the surface. These modified PDMS materials proved also to be biocompatible in cell cultures, which was further confirmed by histological screening of the bladder tissue after implantation in an in vivo pig model.

Cell survival and proliferation after encapsulation in a chemically modified Pluronic(R) F127 hydrogel.

Pluronic® F127 is a biocompatible, injectable, and thermoresponsive polymer with promising biomedical applications. In this study, a chemically modified form, i.e., Pluronic ALA-L with tailored degradation rate, was tested as an encapsulation vehicle for osteoblastic cells. UV cross-linking of the modified polymer results in a stable hydrogel with a slower degradation rate. Toxicological screening showed no adverse effects of the modified Pluronic ALA-L on the cell viability. Moreover, high viability of embedded cells in the cross-linked Pluronic ALA-L was observed with life/death fluorescent staining during a 7-day-culture period. Cells were also cultured on macroporous, cross-linked gelatin microbeads, called CultiSpher-S® carriers, and encapsulated into the modified cross-linked hydrogel. Also, in this situation, good cell proliferation and migration could be observed in vitro. Preliminary in vivo tests have shown the formation of new bone starting from the injected pre-loaded CultiSpher-S® carriers.

Evaluation of bone regeneration with an injectable, in situ polymerizable Pluronic F127 hydrogel derivative combined with autologous mesenchymal stem cells in a goat tibia defect model.

In situ forming bone substitute materials are attractive candidates for filling irregularly shaped defects. In this study, a chemically modified form of the Pluronic F127 hydrogel was used. Similar to the parent form, this derivative underwent a sol-gel transition in the body and additional radical curing resulted in a stable three-dimensional network gel with a controllable degradation rate. An extra cell source of autologous bone marrow-derived mesenchymal stem cells was mixed with the hydrogel to increase the ossification process, when implanted in noncritical size unicortical tibia defects. These cells were cultured and predifferentiated on two types of cell carrier systems, that is, gelatin CultiSpher-S microcarriers and hydroxyapatite tubular carriers. Radiographic and histological evaluation revealed that bone regeneration was comparable in the defects with the bone substitute compositions and the untreated control defects at 2 and 4 weeks postimplantation and that newly formed bone originated from the cells on the CultiSpher-S carriers. This resulted, 6 and 8 weeks postimplantation, in faster bone repair in the defects filled with the hydrogel plus CultiSpher-S carriers in comparison to the control defects. Surprisingly, there was no formation of new bone originating from the hydroxyapatite carriers. The hydrogel by itself seemed to stimulate the natural repair process.

In vitro cytotoxicity testing and the application of elastic interconnection technology for short-term implantable electronics.

An electronic device was fabricated consisting of 2 flexible electronic circuit islands, interconnected by a 7 cm long elastic interconnection, which could be elongated for at least 50%. This interconnection was based on gold conductor tracks following a 2-D spring pattern, embedded in a biocompatible silicone elastomer. The complete device was embedded in the same silicone elastomer. An in vitro cytotoxicity extraction test, executed on small test-samples in accordance with the ISO 10 993-1 guidelines, revealed that the applied silicone encapsulation to these samples functioned as a good seal for at least 8 days.

Evaluation of an injectable, photopolymerizable, and three-dimensional scaffold based on methacrylate-endcapped poly(D,L-lactide-co-epsilon-caprolactone) combined with autologous mesenchymal stem cells in a goat tibial unicortical defect model.

An in situ crosslinkable, biodegradable, methacrylate-endcapped poly(D,L-lactide-co-epsilon-caprolactone) in which crosslinkage is achieved by photoinitiators was developed for bone tissue regeneration. Different combinations of the polymer with bone marrow-derived mesenchymal stem cells (BMSCs) and alpha-tricalcium phosphate (alpha-TCP) were tested in a unicortical tibial defect model in eight goats. The polymers were randomly applied in one of three defects (6.0 mm diameter) using a fourth unfilled defect as control. Biocompatibility and bone-healing characteristics were evaluated by serial radiographies, histology, histomorphometry, and immunohistochemistry. The results demonstrated cell survival and proliferation in the polymer-substituted bone defects. The addition of alpha-TCP was associated with less expansion and growth of the BMSCs than other polymer composites.