An in vitro model to evaluate the properties of matrices produced by fibroblasts from osteogenesis imperfecta and Ehlers-Danlos Syndrome patients.

AIM OF THE STUDY: Osteogenesis imperfecta and Ehlers Danlos syndrome are hereditary disorders caused primarily by defective collagen regulation. Osteogenesis imperfecta patients were divided to haploinsufficient and dominant negative depending on the effect of COL1A1 and COL1A2 mutations whereas Ehlers Danlos syndrome patients had a mutation in PLOD1. Although collagen abnormalities have been extensively studied in monolayer cultures, there are no reports about 3D in vitro models which may reflect more accurately the dynamic cell environment. This is the first study presenting the structural and mechanical characterization of a 3D cell-secreted model using primary patient fibroblasts. MATERIALS AND METHODS: Fibroblasts from patients with osteogenesis imperfecta and Ehlers Danlos syndrome were cultured with ascorbic acid for 5 weeks. The effect of mutations on cytosolic and secreted collagen was tested by electrophoresis following incubation with radiolabeled 14C proline. Extracellular matrix was studied in terms of collagen fiber orientation, stiffness, as well as glycosaminoglycan and collagen content. RESULTS AND CONCLUSIONS: Osteogenesis imperfecta patients with haploinsufficient mutations had higher percentage of anisotropic collagen fibers alignment compared to other patient groups; all patients had a lower percentage of anisotropic samples compared to healthy controls. This correlated with higher average stiffness in the control group. Glycosaminoglycan content was lower in the control and haploinsufficient groups. In cells with PLOD1 mutations, there were no differences in PLOD2 expression. This proof of concept study was able to show differences in collagen fiber orientation between different patient groups which can potentially pave the way towards the development of 3D models aiming at improved investigation of disease mechanisms.

Toward a new generation of pelvic floor implants with electrospun nanofibrous matrices: A feasibility study.

OBJECTIVE: The use of knitted, polypropylene meshes for the surgical treatment of pelvic organ prolapse (POP) is frequently accompanied by severe complications. Looking for alternatives, we studied the potential of three different electrospun matrices in supporting the adhesion, proliferation, and matrix deposition of POP and non-POP fibroblasts, the most important cells to produce extracellular matrix (ECM), in vitro. STUDY DESIGN: We electrospun three commonly used medical materials: nylon; poly (lactide-co-glycolide) blended with poly-caprolactone (PLGA/PCL); and poly-caprolactone blended with gelatin (PCL/Gelatin). The matrices were characterized for their microstructure, hydrophilicity, and mechanical properties. We seeded POP and non-POP fibroblasts from patients with POP and we determined cellular responses and ECM deposition. RESULTS: All matrices had >65% porosity, homogenous microstructures, and close to sufficient tensile strength for pelvic floor repair: 15.4 ± 3.3 MPa for Nylon; 12.4 ± 1.6 MPa for PLGA/PCL; and 3.5 ± 0.9 MPa for PCL/Gelatin. Both the POP and non-POP cells adhered to the electrospun matrices; they proliferated well and produced ample ECM. Overall, the best in vitro performance appeared to be on nylon, presumably because this was the most hydrophilic material with the thinnest fibers. CONCLUSION: Electrospun nanofibrous matrices show feasible mechanical strength and great biocompatibility for POP and non-POP fibroblasts to produce their ECM in vitro and, thus, may be candidates for a new generation of implants for pelvic floor repair. Further studies on electrospun nanofibrous matrices should focus on mechanical and immunological conditions that would be presented in vivo. Neurourol. Urodynam. 36:565-573, 2017. © 2016 Wiley Periodicals, Inc.

Investigation of intervertebral disc degeneration using multivariate FTIR spectroscopic imaging.

Traditionally tissue samples are analysed using protein or enzyme specific stains on serial sections to build up a picture of the distribution of components contained within them. In this study we investigated the potential of multivariate curve resolution-alternating least squares (MCR-ALS) to deconvolute 2nd derivative spectra of Fourier transform infrared (FTIR) microscopic images measured in transflectance mode of goat and human paraffin embedded intervertebral disc (IVD) tissue sections, to see if this methodology can provide analogous information to that provided by immunohistochemical stains and bioassays but from a single section. MCR-ALS analysis of non-degenerate and enzymatically in vivo degenerated goat IVDs reveals five matrix components displaying distribution maps matching histological stains for collagen, elastin and proteoglycan (PG), as well as immunohistochemical stains for collagen type I and II. Interestingly, two components exhibiting characteristic spectral and distribution profiles of proteoglycans were found, and relative component/tissue maps of these components (labelled PG1 and PG2) showed distinct distributions in non-degenerate versus mildly degenerate goat samples. MCR-ALS analysis of human IVD sections resulted in comparable spectral profiles to those observed in the goat samples, highlighting the inter species transferability of the presented methodology. Multivariate FTIR image analysis of a set of 43 goat IVD sections allowed the extraction of semi-quantitative information from component/tissue gradients taken across the IVD width of collagen type I, collagen type II, PG1 and PG2. Regional component/tissue parameters were calculated and significant correlations were found between histological grades of degeneration and PG parameters (PG1: p = 0.0003, PG2: p < 0.0001); glycosaminoglycan (GAG) content and PGs (PG1: p = 0.0055, PG2: p = 0.0001); and MRI T2* measurements and PGs (PG1: p = 0.0021, PG2: p < 0.0001). Additionally, component/tissue parameters for collagen type I and II showed significant correlations with total collagen content (p = 0.0204, p = 0.0127). In conclusion, the presented findings illustrate, that the described multivariate FTIR imaging approach affords the necessary chemical specificity to be considered an important tool in the study of IVD degeneration in goat and human IVDs.

Spinal fusion using adipose stem cells seeded on a radiolucent cage filler: a feasibility study of a single surgical procedure in goats.

PURPOSE: To assess the feasibility of a one-step surgical concept, employing adipose stem cells (ASCs) and a novel degradable radiolucent cage filler (poly-L-lactide-co-caprolactone; PLCL), within polyetheretherketone cages in a stand-alone caprine spinal fusion model. METHODS: A double-level fusion study was performed in 36 goats. Four cage filler groups were defined: (i) acellular PLCL, (ii) PLCL + SVF (freshly harvested stromal vascular fraction highly enriched in ASCs); (iii) PLCL + ASCs (cultured to homogeneity); and (iv) autologous iliac crest bone graft (ABG). Fusion was assessed after 3 and 6 months by radiography, micro-CT, biomechanics, and biochemical analysis of tissue formed inside the cage after 6 months. RESULTS: No adverse effects were observed in all groups. After 3 months, similar and low fusion rates were found. Segmental stability did not differ between groups in all tested directions. Micro-CT imaging revealed significantly higher amounts of mineralized tissue in the ABG group compared to all others. After 6 months, interbody fusion rates were: PLCL 53%, SVF 30%, ASC 43% and ABG 63%. A trend towards higher mineralized tissue content was found for the ABG group. Biochemical and biomechanical analyses revealed equal maturity of collagen cross-links and similar segmental stability between all groups. CONCLUSIONS: This study demonstrates the technical feasibility and safety of the one-step surgical procedure for spinal fusion for the first time. The radiolucent PLCL scaffold allowed in vivo monitoring of bone formation using plain radiography. Addition of stem cells to the PLCL scaffolds did not result in adverse effects, but did not enhance the rate and number of interbody fusions under the current conditions. A trend towards superior results with ABG was found. Further research is warranted to optimize the spinal fusion model for proper evaluation of both PLCL and stem cell therapy.

MRI T2* mapping correlates with biochemistry and histology in intervertebral disc degeneration in a large animal model.

PURPOSE: To evaluate intervertebral disc (IVD) degeneration and treatments, an objective diagnostic tool is needed. Recently, T2* relaxation time mapping was proposed as a technique to assess early IVD degeneration, yet the correlation with biochemical content and histological features has not been investigated previously. Our objective was to validate T2* mapping for disc degeneration by correlating this technique with accepted parameters of IVD degeneration. METHODS: Mildly and severely degenerated lumbar discs were obtained from an in vivo large animal study; two healthy goat spines were acquired as control. In total, 48 IVDs were analysed using T2-weighted MRI, T2* relaxation time mapping, biochemical assays, macroscopic and histological scoring. Correlations between variables were expressed with Spearman's rho (ρ) coefficients. RESULTS: A complete range of degenerative grades were obtained (mean histological grade 2.2, range 0-6). A linear positive correlation was observed between T2* relaxation time and glycosaminoglycan content (ρ = 0.64, p < 0.001). T2* relaxation time decreased linearly with increasing degeneration as assessed with Pfirrmann scoring system (ρ = -0.67, p < 0.001), macroscopic (ρ = -0.33, p < 0.05) and histological (ρ = -0.45, p < 0.05) grading. CONCLUSIONS: T2* mapping is an MRI technique for IVD evaluation which allows for measurements on a continuous scale thus minimising observer bias compared to grading systems. Although limited by a small sample size, this study showed a relatively good and linear correlation between T2* relaxation time and accepted parameters of disc degeneration. This suggests that T2* mapping is a promising tool to assess disc degeneration in clinical practice.

Functional characteristics of vaginal fibroblastic cells from premenopausal women with pelvic organ prolapse.

Pelvic organ prolapse (POP) remains a great therapeutic challenge with no optimal treatment available. Tissue maintenance and remodelling are performed by fibroblasts, therefore altered cellular functionality may influence tissue quality. In this study, we evaluated functional characteristics of fibroblastic cells from tissues involved in POP. To rule out normal ageing tissue degeneration, biopsies from 18 premenopausal women were collected from the precervical region (non-POP site) after hysterectomy of 8 healthy and 10 POP cystocele cases (POP-Q stage ≥ II). Extra tissues from the prolapsed sites were taken in the POP cases to distinguish between intrinsic and acquired cellular defects. Twenty-eight primary fibroblastic cultures were studied in vitro. A contractility assay was used to test fibroblast-mediated collagen contraction. Cellular mechanoresponses on collagen-coated or uncoated substrates were evaluated by measuring matrix remodelling factors at protein or gene expression levels. No differences were found between fibroblasts from the controls and the non-POP site of the case group. Fibroblastic cells from the prolapsed site showed delayed fibroblast-mediated collagen contraction and lower production of matrix metalloproteinase-2 (MMP-2) on collagen-coated plates. On uncoated surfaces the gene MMP-2 and its tissue inhibitor of metalloproteinases-2 were up-regulated in POP site fibroblastic cells. In conclusion, fibroblastic cells derived from prolapsed tissues of patients with cystocele, display altered in vitro functional characteristics depending on the surface substrate and compared with non-prolapsed site. This implies an acquired rather than an intrinsic defect for most patients with cystocele, and should be taken into account when trying to improve treatments for POP.

Intradiscal pressure depends on recent loading and correlates with disc height and compressive stiffness.

PURPOSE: Intervertebral discs exhibit time-dependent deformation (creep), which could influence the relation between applied stress and intradiscal pressure. This study investigates the effect of prolonged dynamic loading on intradiscal pressure, disc height and compressive stiffness, and examines their mutual relationships. METHODS: Fifteen caprine lumbar discs with 5 mm of vertebral bone on either side were compressed by 1 Hz sinusoidal load for 4.5 h. After preload, 'High' (130 ± 20 N) or 'Low' (50 ± 10 N) loads were alternated every half hour. Continuous intradiscal pressure measurement was performed with a pressure transducer needle. RESULTS: Each disc showed a linear relationship between axial compression and intradiscal pressure (R (2) > 0.91). The intercept of linear regression analysis declined over time, but the gradient remained constant. Disc height changes were correlated to intradiscal pressure changes (R (2) > 0.98): both decreased during High loading, and increased during Low loading. In contrast, compressive stiffness increased during High loading, and was inversely related to intradiscal pressure and disc height. CONCLUSIONS: Intradiscal pressure is influenced by recent loading due to fluid flow. The correlations found in this study suggest that intradiscal pressure is important for disc height and axial compliance. These findings are relevant for mechanobiology studies, nucleus replacements, finite element models, and ex vivo organ culture systems.

A three-dimensional algorithm for precise measurement of human auricle parameters.

Measurement of auricle parameters for planning and post-operative evaluation presents substantial challenges due to the complex 3D structure of the human auricle. Traditional measurement methods rely on manual techniques, resulting in limited precision. This study introduces a novel automated surface-based three-dimensional measurement method for quantifying human auricle parameters. The method was applied to virtual auricles reconstructed from Computed Tomography (CT) scans of a cadaver head and subsequent measurement of important clinically relevant aesthetical auricular parameters (length, width, protrusion, position, auriculocephalic angle, and inclination angle). Reference measurements were done manually (using a caliper and using a 3D landmarking method) and measurement precision was compared to the automated method. The CT scans were performed using both a contemporary high-end and a low-end CT scanner. Scans were conducted at a standard scanning dose, and at half the dose. The automatic method demonstrated significantly higher precision in measuring auricle parameters compared to manual methods. Compared to traditional manual measurements, precision improved for auricle length (9×), width (5×), protrusion (5×), Auriculocephalic Angle (5-54×) and posteroanterior position (23×). Concerning parameters without comparison with a manual method, the precision level of supero-inferior position was 0.489 mm; and the precisions of the inclination angle measurements were 1.365 mm and 0.237 mm for the two automated methods investigated. Improved precision of measuring auricle parameters was associated with using the high-end scanner. A higher dose was only associated with a higher precision for the left auricle length. The findings of this study emphasize the advantage of automated surface-based auricle measurements, showcasing improved precision compared to traditional methods. This novel algorithm has the potential to enhance auricle reconstruction and other applications in plastic surgery, offering a promising avenue for future research and clinical application.

Fibroblasts from women with pelvic organ prolapse show differential mechanoresponses depending on surface substrates.

INTRODUCTION AND HYPOTHESIS: Little is known about dynamic cell-matrix interactions in the context of pathophysiology and treatments for pelvic organ prolapse (POP). This study sought to identify differences between fibroblasts from women with varying degrees of prolapse in reaction to mechanical stimuli and matrix substrates in vitro. METHODS: Fibroblasts from the vaginal wall of three patients with POP Quantification (POP-Q) system stages 0, II, and IV were stretched on artificial polymer substrates either coated or not coated with collagen I. Changes in morphology and anabolic/catabolic compounds that affect matrix remodelling were evaluated at protein- and gene-expression levels. Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Tukey-Kramer's post hoc test. RESULTS: POP fibroblasts show delayed cell alignment and lower responses to extracellular matrix remodelling factors at both enzymatic- and gene-expression levels compared with healthy fibroblasts. CONCLUSION: POP fibroblasts, when compared with healthy cells, show differential mechanoresponses on two artificial polymer substrates. This should be taken into account when designing or improving implants for treating POP.

Tissue Engineering Neovagina for Vaginoplasty in Mayer-Rokitansky-Küster-Hauser Syndrome and Gender Dysphoria Patients: A Systematic Review.

Background: Vaginoplasty is a surgical solution to multiple disorders, including Mayer-Rokitansky-Küster-Hauser syndrome and male-to-female gender dysphoria. Using nonvaginal tissues for these reconstructions is associated with many complications, and autologous vaginal tissue may not be sufficient. The potential of tissue engineering for vaginoplasty was studied through a systematic bibliography search. Cell types, biomaterials, and signaling factors were analyzed by investigating advantages, disadvantages, complications, and research quantity. Search Methods: A systematic search was performed in Medline, EMBASE, Web of Science, and Scopus until March 8, 2022. Term combinations for tissue engineering, guided tissue regeneration, regenerative medicine, and tissue scaffold were applied, together with vaginoplasty and neovagina. The snowball method was performed on references and a Google Scholar search on the first 200 hits. Original research articles on human and/or animal subjects that met the inclusion (reconstruction of vaginal tissue and tissue engineering method) and no exclusion criteria (not available as full text; written in foreign language; nonoriginal study article; genital surgery other than neovaginal reconstruction; and vaginal reconstruction with autologous or allogenic tissue without tissue engineering or scaffold) were assessed. The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) checklist, the Newcastle-Ottawa Scale, and the Gold Standard Publication Checklist were used to evaluate article quality and bias. Outcomes: A total of 31 out of 1569 articles were included. Data extraction was based on cell origin and type, biomaterial nature and composition, host species, number of hosts and controls, neovaginal size, replacement fraction, and signaling factors. An overview of used tissue engineering methods for neovaginal formation was created, showing high variance of cell types, biomaterials, and signaling factors and the same topics were rarely covered multiple times. Autologous vaginal cells and extracellular matrix-based biomaterials showed preferential properties, and stem cells carry potential. However, quality confirmation of orthotopic cell-seeded acellular vaginal matrix by clinical trials is needed as well as exploration of signaling factors for vaginoplasty. Impact statement General article quality was weak to sufficient due to unreported cofounders and incomplete animal study descriptions. Article quality and heterogenicity made identification of optimal cell types, biomaterials, or signaling factors unreliable. However, trends showed that autologous cells prevent complications and compatibility issues such as healthy cell destruction, whereas stem cells prevent cross talk (interference of signaling pathways by signals from other cell types) and rejection (but need confirmation testing beyond animal trials). Natural (orthotopic) extracellular matrix biomaterials have great preferential properties that encourage future research, and signaling factors for vascularization are important for tissue engineering of full-sized neovagina.

Injectable biomaterial induces regeneration of the intervertebral disc in a caprine loaded disc culture model.

Back pain is the leading cause of disability with half of cases attributed to intervertebral disc (IVD) degeneration, yet currently no therapies target this cause. We previously reported an ex vivo caprine loaded disc culture system (LDCS) that accurately represents the cellular phenotype and biomechanical environment of human IVD degeneration. Here, the efficacy of an injectable hydrogel system (LAPONITE® crosslinked pNIPAM-co-DMAc, (NPgel)) to halt or reverse the catabolic processes of IVD degeneration was investigated within the LDCS. Following enzymatic induction of degeneration using 1 mg mL-1 collagenase and 2 U mL-1 chondroitinase ABC within the LDCS for 7 days, IVDs were injected with NPgel alone or with encapsulated human bone marrow progenitor cells (BMPCs). Un-injected caprine discs served as degenerate controls. IVDs were cultured for a further 21 days within the LDCS. Tissues were then processed for histology and immunohistochemistry. No extrusion of NPgel was observed during culture. A significant decrease in histological grade of degeneration was seen in both IVDs injected with NPgel alone and NPgel seeded with BMPCs, compared to un-injected controls. Fissures within degenerate tissue were filled by NPgel and there was evidence of native cell migration into injected NPgel. The expression of healthy NP matrix markers (collagen type II and aggrecan) was increased, whereas the expression of catabolic proteins (MMP3, ADAMTS4, IL-1ß and IL-8) was decreased in NPgel (±BMPCs) injected discs, compared to degenerate controls. This demonstrates that NPgel promotes new matrix production at the same time as halting the degenerative cascade within a physiologically relevant testing platform. This highlights the potential of NPgel as a future therapy for IVD degeneration.

Objective quantitative methods to evaluate microtia reconstruction: A scoping review.

Background: Commonly used methods to evaluate auricles are subjective and are therefore not specific, comprehensive, and precise nor effective in the assessment of microtia reconstruction outcomes. This scoping review aimed to summarize the objective methods for the accurate evaluation of microtia reconstruction. Methods: We performed a scoping review of publications that used objective measurement methods to evaluate outcomes of microtia reconstruction according to the PRISMA-ScR guidelines. A systematic literature search was conducted in the Embase, PubMed, Cochrane, CNKI, and VIP databases, and literature references were screened for additional records. Studies that evaluated auricles after microtia reconstruction using quantitative anthropometric methods were included, and data on these methods were collected. Results: Twenty-five publications reported on quantitative objective outcome measurements. Thirteen studies evaluated auricular protrusion, three articles assessed the position or symmetry, and twelve studies reported on auricle size. The quantitative measurements of fine structures, such as the tragus and concha, were described in three studies. All described measurements used manual landmarking, where fifteen studies described well-defined landmarks, fifteen studies described poorly defined landmarks, and four studies used a combination of well and poorly defined landmarks. Conclusion: The objective evaluation of microtia reconstruction outcomes is hindered by significant heterogeneity of measurement methods. The measurement methods used for general auricular measurements (auricular protrusion, auriculocephalic angle, and size) used in microtia reconstruction were abundant, while measurements of auricular position and the fine structures of the auricle were limited. Three-dimensional imaging combined with computer analyses poses promising future alternatives.

Cyclic mechanical stretch reduces myofibroblast differentiation of primary lung fibroblasts.

In lung fibrosis tissue architecture and function is severely hampered by myofibroblasts due to excessive deposition of extracellular matrix and tissue contraction. Myofibroblasts differentiate from fibroblasts under the influence of transforming growth factor (TGF) ß(1) but this process is also controlled mechanically by cytoskeletal tension. In healthy lungs, the cytoskeleton of fibroblasts is mechanically strained during breathing. In stiffer fibrotic lung tissue, this mechanical stimulus is reduced, which may influence fibroblast-to-myofibroblast differentiation. Therefore, we investigated the effect of cyclic mechanical stretch on fibroblast-to-myofibroblast differentiation. Primary normal human lung fibroblasts were grown on BioFlex culture plates and stimulated to undergo myofibroblast differentiation by 10 ng/ml TGFß(1). Cells were either or not subjected to cyclic mechanical stretch (sinusoidal pattern, maximum elongation 10%, 0.2 Hz) for a period of 48 h on a Flexercell apparatus. mRNA expression was analyzed by real-time PCR. Cyclic mechanical loading reduced the mRNA expression of the myofibroblast marker α-smooth muscle actin and the extracellular matrix proteins type-I, type-III, and type-V collagen, and tenascin C. These outcomes indicate that fibroblast-to-myofibroblast differentiation is reduced. Cyclic mechanical loading did not change the expression of the fibronectin ED-A splice variant, but did decrease the paracrine expression of TGFß(1), thereby suggesting a possible regulation mechanism for the observed effects. The data suggest that cyclic loading experienced by healthy lung cells during breathing may prevent fibroblasts from differentiating towards myofibroblasts.

A Single Axial Impact Load Causes Articular Damage That Is Not Visible with Micro-Computed Tomography: An Ex Vivo Study on Caprine Tibiotalar Joints.

OBJECTIVE: Excessive articular loading, for example, an ankle sprain, may result in focal osteochondral damage, initiating a vicious degenerative process resulting in posttraumatic osteoarthritis (PTOA). Better understanding of this degenerative process would allow improving posttraumatic care with the aim to prevent PTOA. The primary objective of this study was to establish a drop-weight impact testing model with controllable, reproducible and quantitative axial impact loads to induce osteochondral damage in caprine tibiotalar joints. We aimed to induce osteochondral damage on microscale level of the tibiotalar joint without gross intra-articular fractures of the tibial plafond. DESIGN: Fresh-frozen tibiotalar joints of mature goats were used as ex vivo articulating joint models. Specimens were axially impacted by a mass of 10.5 kg dropped from a height of 0.3 m, resulting in a speed of 2.4 m/s, an impact energy of 31.1 J and an impact impulse of 25.6 N·s. Potential osteochondral damage of the caprine tibiotalar joints was assessed using contrast-enhanced high-resolution micro-computed tomography (micro-CT). Subsequently, we performed quasi-static loading experiments to determine postimpact mechanical behavior of the tibiotalar joints. RESULTS: Single axial impact loads with a mass of 15.5 kg dropped from 0.3 m, resulted in intra-articular fractures of the tibial plafond, where a mass of 10.55 kg dropped from 0.3 m did not result in any macroscopic damage. In addition, contrast-enhanced high-resolution micro-CT imaging neither reveal any acute microdamage (i.e., microcracks) of the subchondral bone nor any (micro)structural changes in articular cartilage. The Hexabrix content or voxel density (i.e., proteoglycan content of the articular cartilage) on micro-CT did not show any differences between intact and impacted specimens. However, quasi-static whole-tibiotalar-joint loading showed an altered biomechanical behavior after application of a single axial impact (i.e., increased hysteresis when compared with the intact or nonimpacted specimens). CONCLUSIONS: Single axial impact loads did not induce osteochondral damage visible with high-resolution contrast-enhanced micro-CT. However, despite the lack of damage on macro- and even microscale, the single axial impact loads resulted in "invisible injuries" because of the observed changes in the whole-joint biomechanics of the caprine tibiotalar joints. Future research must focus on diagnostic tools for the detection of early changes in articular cartilage after a traumatic impact (i.e., ankle sprains or ankle fractures), as it is well known that this could result in PTOA.

Inorganic phosphate stimulates DMP1 expression in human periodontal ligament fibroblasts embedded in three-dimensional collagen gels.

Stable integration of collagenous tissue-engineered constructs to surrounding solid devices can be accomplished by coating the solid surfaces with exogenous alkaline phosphatase (ALP). We showed previously that coating of culture well surfaces with the enzyme in combination with the presence of its substrate beta-glycerophosphate (beta-GP) induces mineral deposition at the interface of matrix and surface, thereby preventing matrix detachment. In this study the effect of such mineral-inducing conditions on differentiation of human periodontal ligament (PDL) fibroblasts into osteoblasts/cementoblasts was analyzed in three-dimensional collagen gels. Mineral-inducing conditions decreased collagen type I gene expression and induced dentin matrix protein 1 (DMP1; a marker of late osteoblasts/cementoblasts) gene expression by fibroblasts. DMP1 protein was detected in some fibroblasts only in mineralizing gels. Exogenous ALP released high levels of inorganic phosphate from beta-GP. Addition of inorganic phosphate alone induced DMP1 gene expression, which could be prevented by blocking phosphate entry into fibroblasts by foscarnet. We concluded that mineralizing conditions induced by exogenous ALP affect the phenotype of PDL fibroblasts. The fibroblasts are stimulated to express the late osteoblast/osteocyte marker protein DMP1, which is mediated by uptake of inorganic phosphate into the cells. The enzyme-mediated mineral deposition may thus facilitate enhanced integration of collagenous tissue-engineered constructs to devices or implants in vitro.

Time-dependent failure of amorphous polylactides in static loading conditions.

Polylactides are commonly praised for their excellent mechanical properties (e.g. a high modulus and yield strength). In combination with their bioresorbability and biocompatibility, they are considered prime candidates for application in load-bearing biomedical implants. Unfortunately, however, their long-term performance under static load is far from impressive. In a previous in vivo study on degradable polylactide spinal cages in a goat model it was observed that, although short-term mechanical and real-time degradation experiments predicted otherwise, the implants failed prematurely under the specified loads. In this study we demonstrate that this premature failure is attributed to the time-dependent character of the material used. The phenomenon is common to all polymers, and finds its origin in stress-activated segmental molecular mobility leading to a steady rate of plastic flow. The stress-dependence of this flow-rate is well captured by Eyring's theory of absolute rates, as demonstrated on three amorphous polylactides of different stereoregularity.We show that the kinetics of the three materials are comparable and can be well described using the proposed modeling framework. The main conclusion is that knowledge of the instantaneous strength of a polymeric material is insufficient to predict its long-term performance.

Collagen type V modulates fibroblast behavior dependent on substrate stiffness.

Collagen type V is highly expressed during tissue development and wound repair, but its exact function remains unclear. Cell binding to collagen V affects various basic cell functions and increased collagen V levels alter the structural organization and the stiffness of the ECM. We studied the combined effects of collagen V and substrate stiffness on the morphology, focal adhesion formation, and actin organization of fibroblasts. We found that a hybrid collagen I/V coating impairs fibroblast spreading on soft substrates (<10 kPa), but not on stiffer substrates (68 kPa or glass). In sharp contrast, a pure collagen I coating does not impair cell spreading on soft substrates. The impairment of cell spreading by collagen V is accompanied by diffuse actin staining patterns and small focal adhesions. These observations suggest that collagen V plays an essential role in modifying cell behavior during development and remodeling, when very soft tissues are present.

Noise enhances the rapid nitric oxide production by bone cells in response to fluid shear stress.

Stochastic resonance is exhibited by many biological systems, where the response to a small stimulus is enhanced with the aid of noise. This intriguing possibility provides a novel paradigm for understanding previously reported osteogenic benefits of low amplitude dynamic loading. However, it is unknown whether bone cell mechanosensitivity is enhanced by noise as an alternative mechanism for an amplified response to small stresses. We studied whether noise of varying intensities enhanced the mechanosensitivity of MC3T3-E1 cells. Nitric oxide (NO) production was measured as the parameter for bone cell activation. Dynamic fluid shear stress stimulated bone cells provided an initial-stress kick was implemented. Without the initial stress-kick bone cells did not release a significant amount of NO demonstrating an essential non-linearity to bone cell responses to stress and the possibility of stochastic resonance in bone cell mechanosensitivity. The rapid NO response of MC3T3-E1 cells to a small periodic fluid shear stress was increased with the addition of noise compared to the response to stress with only noise. This confirms the possibility of stochastic resonance enhancement of NO production by bone cells. Since NO regulate bone formation as well as resorption, our results suggest that noise enhances the activity of bone cells in driving the mechanical adaptation of bone.

A salt-based method to adapt stiffness and biodegradability of porous collagen scaffolds.

Type I collagen scaffolds for tissue reconstruction often have impaired mechanical characteristics such as limited stiffness and lack of strength. In this study, a new technique is presented to fine-tune stiffness and biodegradability of collagen scaffolds by treatment with concentrated salt solutions. Collagen scaffolds were prepared by a casting, freezing and lyophilization process. Scaffolds were treated with 90% saturated salt solutions, the salts taken from the Hofmeister series, followed by chemical crosslinking. Treatment with salts consisting of a divalent cation in combination with a monovalent anion, e.g. CaCl2, resulted in fast shrinkage of the scaffolds up to approximately 10% of the original surface area. Effective salts were mostly at the chaotropic end of the Hofmeister series. Shrunken scaffolds were more than 10 times stiffer than non-shrunken control scaffolds, and displayed reduced pore sizes and swollen, less organized collagen fibrils. The effect could be pinpointed to the level of individual collagen molecules and indicates the shrinking effect to be driven by disruption of stabilizing hydrogen bonds within the triple helix. No calcium deposits remained in CaCl2 treated scaffolds. Subcutaneous implantation in rats showed similar biocompatibility compared to H2O and NaCl treated scaffolds, but reduced cellular influx and increased structural integrity without signs of major degradation after 3 months. In conclusion, high concentrations of chaotropic salts can be used to adjust the mechanical characteristics of collagen scaffolds without affecting biocompatibility. This technique may be used in regenerative medicine to stiffen collagen scaffolds to better comply with the surrounding tissues, but may also be applied for e.g. slow release drug delivery systems.

Osteocyte morphology in fibula and calvaria --- is there a role for mechanosensing?

INTRODUCTION: External mechanical forces on cells are known to influence cytoskeletal structure and thus cell shape. Mechanical loading in long bones is unidirectional along their long axes, whereas the calvariae are loaded at much lower amplitudes in different directions. We hypothesised that if osteocytes, the putative bone mechanosensors, can indeed sense matrix strains directly via their cytoskeleton, the 3D shape and the long axes of osteocytes in fibulae and calvariae will bear alignment to the different mechanical loading patterns in the two types of bone. MATERIALS AND METHODS: We used confocal laser scanning microscopy and nano-computed tomography to quantitatively determine the 3D morphology and alignment of long axes of osteocytes and osteocyte lacunae in situ. RESULTS: Fibular osteocytes showed a relatively elongated morphology (ratio lengths 5.9:1.5:1), whereas calvarial osteocytes were relatively spherical (ratio lengths 2.1:1.3:1). Osteocyte lacunae in fibulae had higher unidirectional alignment than the osteocyte lacunae in calvariae as demonstrated by their degree of anisotropy (3.33 and 2.10, respectively). The long axes of osteocyte lacunae in fibulae were aligned parallel to the principle mechanical loading direction, whereas those of calvarial osteocyte lacunae were not aligned in any particular direction. CONCLUSIONS: The anisotropy of osteocytes and their alignment to the local mechanical loading condition suggest that these cells are able to directly sense matrix strains due to external loading of bone. This reinforces the widely accepted role of osteocytes as mechanosensors, and suggests an additional mode of mechanosensing besides interstitial fluid flow. The relatively spherical morphology of calvarial osteocytes suggests that these cells are more mechanosensitive than fibular osteocytes, which provides a possible explanation of efficient physiological load bearing for the maintenance of calvarial bone despite its condition of relative mechanical disuse.