Collagen-rich Connective Tissue Research Articles

Alkaptonuria.

Alkaptonuria is a rare inborn error of metabolism caused by the deficiency of homogentisate 1,2-dioxygenase activity. The consequent homogentisic acid (HGA) accumulation in body fluids and tissues leads to a multisystemic and highly debilitating disease whose main features are dark urine, ochronosis (HGA-derived pigment in collagen-rich connective tissues), and a painful and severe form of osteoarthropathy. Other clinical manifestations are extremely variable and include kidney and prostate stones, aortic stenosis, bone fractures, and tendon, ligament and/or muscle ruptures. As an autosomal recessive disorder, alkaptonuria affects men and women equally. Debilitating symptoms appear around the third decade of life, but a proper and timely diagnosis is often delayed due to their non-specific nature and a lack of knowledge among physicians. In later stages, patients' quality of life might be seriously compromised and further complicated by comorbidities. Thus, appropriate management of alkaptonuria requires a multidisciplinary approach, and periodic clinical evaluation is advised to monitor disease progression, complications and/or comorbidities, and to enable prompt intervention. Treatment options are patient-tailored and include a combination of medications, physical therapy and surgery. Current basic and clinical research focuses on improving patient management and developing innovative therapies and implementing precision medicine strategies.

Fibromatous Epulis: A Clinical Case Report

A 35-year-old patient presented with a painless, broad-based exophytic lesion in the buccal interdental region between teeth 13 and 14. Despite oral hygiene efforts the lesion persisted for around one year. Radiology excluded bone involvement, and histopathology after excision confirmed a fibromatous epulis, which is characterized by collagen-rich connective tissue. There was no recurrence within one-year follow-up. Surgical removal proved to be efficient.

Dynamic changes in mitral valve extracellular matrix, tissue mechanics and function in a mouse model of Marfan syndrome

ObjectiveMouse models of Marfan syndrome (MFS) with Fibrillin 1 (Fbn1) variant C1041G exhibit cardiovascular abnormalities, including myxomatous valve disease (MVD) and aortic aneurism, with structural extracellular matrix (ECM) dysregulation. In this study, we examine the structure-function-mechanics relations of the mitral valve related to specific transitions in ECM composition and organization in progressive MVD in MFS mice from Postnatal day (P)7 to 1 year-of-age. Approach and resultsMechanistic links between mechanical forces and biological changes in MVD progression were examined in Fbn1C1041G/+ MFS mice. By echocardiography, mitral valve dysfunction is prevalent at 2 months with a decrease in cardiac function at 6 months, followed by a preserved cardiac function at 12 months. Mitral valve (MV) regurgitation occurs in a subset of mice at 2–6 months, while progressive dilatation of the aorta occurs from 2 to 12 months. Mitral valve tissue mechanical assessments using a uniaxial Permeabilizable Fiber System demonstrate decreased stiffness of MFS MVs at all stages. Histological and microscopic analysis of ECM content, structure, and fiber orientation demonstrate that alterations in ECM mechanics, composition, and organization precede functional abnormalities in Fbn1C1041G/+MFS MVs. At 2 months, ECM abnormalities are detected with an increase in proteoglycans and decreased stiffness of the mitral valve. By 6–12 months, collagen fiber remodeling is increased with abnormal fiber organization in MFS mitral valve leaflets. At the same time, matrifibrocyte gene expression characteristic of collagen-rich connective tissue is increased, as detected by RNA in situ hybridization and qPCR. Together, these studies demonstrate early prevalence of proteoglycans at 2 months followed by upregulation of collagen structure and organization with age in MVs of MFS mice. ConclusionsAltogether, our data indicate dynamic regulation of mitral valve structure, tissue mechanics, and function that reflect changes in ECM composition, organization, and gene expression in progressive MVD. Notably, increased collagen fiber organization and orientation, potentially dependent on increased matrifibrocyte cell activity, is apparent with altered mitral valve mechanics and function in aging MFS mice.

SLIT3-mediated fibroblast signaling: a promising target for antifibrotic therapies.

The SLIT family (SLIT1-3) of highly conserved glycoproteins was originally identified as ligands for the Roundabout (ROBO) family of single-pass transmembrane receptors, serving to provide repulsive axon guidance cues in the nervous system. Intriguingly, studies involving SLIT3 mutant mice suggest that SLIT3 might have crucial biological functions outside the neural context. Although these mutant mice display no noticeable neurological abnormalities, they present pronounced connective tissue defects, including congenital central diaphragmatic hernia, membranous ventricular septal defect, and osteopenia. We recently hypothesized that the phenotype observed in SLIT3-deficient mice may be tied to abnormalities in fibrillar collagen-rich connective tissue. Further research by our group indicates that both SLIT3 and its primary receptor, ROBO1, are expressed in fibrillar collagen-producing cells across various nonneural tissues. Global and constitutive SLIT3 deficiency not only reduces the synthesis and content of fibrillar collagen in various organs but also alleviates pressure overload-induced fibrosis in both the left and right ventricles. This review delves into the known phenotypes of SLIT3 mutants and the debated role of SLIT3 in vasculature and bone. Present evidence hints at SLIT3 acting as an autocrine regulator of fibrillar collagen synthesis, suggesting it as a potential antifibrotic treatment. However, the precise pathway and mechanisms through which SLIT3 regulates fibrillar collagen synthesis remain uncertain, presenting an intriguing avenue for future research.

Contact Guidance of Connective Tissue Fibroblasts on Submicrometer Anisotropic Topographical Cues Is Dependent on Tissue of Origin, β1 Integrins, and Tensin-1 Recruitment.

The substratum topography of both natural and synthetic materials is a prominent regulator of cell behaviors including adhesion, migration, matrix fibrillogenesis, and cell phenotype. Connective tissue fibroblasts are known to respond to repeating groove topographical modifications by aligning and exhibiting directed migration, a phenomenon termed contact guidance. Although both reside in collagen rich connective tissues, dermal and gingival fibroblasts are known to exhibit differences in phenotype during wound healing, with gingival tissue showing a fetal-like scarless response. Differences in adhesion formation and maturation are known to underlie both a scarring phenotype and cell response to topographical features. Utilizing repeating groove substrates with periodicities of 600, 900, and 1200 nm (depth, 100 nm), we investigated the roles of integrins αvβ3 and β1 associated adhesions on contact guidance of human gingival (HGFs) and dermal fibroblasts (HDFs). HGFs showed a higher degree of orientation with the groove long axis than HDFs, with alignment of both vinculin and tensin-1 evident on 600 and 900 nm periodicities in both cell types. Orientation with grooves of any periodicity in HGFs and HDFs did not alter the adhesion number or area compared to smooth control surfaces. Growth of both cell types on all periodicities reduced fibronectin fibrillogenesis compared to control surfaces. Independent inhibition of integrin αvβ3 and β1 in both cell types induced changes in spreading up to 6 h and reduced alignment with the groove long axis. At 24 h post-seeding with blocking antibodies, HGFs recovered orientation, but in HDFs, blocking of β1, but not αvβ3 integrins, inhibited alignment. Blocking of β1 and αvβ3 in HDFs, but not HGFs, inhibited tensin-1-associated fibrillar adhesion formation. Furthermore, inhibition of β1 integrins in HDFs, but not HGFs, resulted in recruitment of tensin-1 to αvβ3 focal adhesions, preventing HDFs from aligning with the groove long axis. Our work demonstrates that tensin-1 localization with specific integrins in adhesion sites is an important determinant of contact guidance. This work emphasizes further the need for tissue-specific biomaterials, when integration into host tissues is required.

Three-dimensional remodeling of collagen fibers within cervical tissues in pregnancy

The cervix is a collagen-rich connective tissue that must remain closed during pregnancy while undergoing progressive remodeling in preparation for delivery, which begins before the onset of the preterm labor process. Therefore, it is important to resolve the changes of collagen fibers during cervical remodeling for the prevention of preterm labor. Herein, we assessed the spatial organization of collagen fibers in a three-dimensional (3D) context within cervical tissues of mice on day 3, 9, 12, 15 and 18 of gestation. We found that the 3D directional variance, a novel metric of alignment, was higher on day 9 than that on day 3 and then gradually decreased from day 9 to day 18. Compared with two-dimensional (2D) approach, a higher sensitivity was achieved from 3D analysis, highlighting the importance of truly 3D quantification. Moreover, the depth-dependent variation of 3D directional variance was investigated. By combining multiple 3D directional variance-derived metrics, a high level of classification accuracy was acquired in distinguishing different periods of pregnancy. These results demonstrate that 3D directional variance is sensitive to remodeling of collagen fibers within cervical tissues, shedding new light on highly-sensitive, early detection of preterm birth (PTB).

Assessment of Pseudocoarctation of the Aorta with Saccular Aneurysms by Four-Dimensional Flow Magnetic Resonance Imaging and Histological Analysis.

In this study, we present the case of a 21-year-old woman with pseudocoarctation of the aorta with saccular aneurysms that were evaluated by four-dimensional flow magnetic resonance imaging and histological analysis. We observed complete occupation of the aneurysm sacs by vortex flow and high peak wall shear stress in the proximal region of the kinked aorta. The aortic replacement was performed for the thoracic aortic aneurysms and the clinical course was uneventful. The aneurysms were histopathologically diagnosed as pseudoaneurysms based on the disappearance of all three layers and their replacement with collagen-rich connective tissues. These findings indicate that abnormal flow dynamics and the resulting abnormal shear stress in the aorta may play central roles in the formation and development of a saccular aneurysm.

Strontium-loaded hydrogel scaffolds to promote gingival fibroblast function.

Dental implant clinical success is dependent on effective peri-implant tissue attachment to the trans-mucosal portion following placement. Modification of transmucosal implant surfaces can improve cellular adhesion and function leading to formation of an effective soft-tissue seal during healing, of which gingival fibroblasts are prominent cells to migrate to repair wounds and crucial for the development of a collagen rich connective tissue. Biocompatible loaded scaffold materials have been developed to allow local release of molecules with effective biological activity. Our previous studies indicate that strontium can promote gingival fibroblast metabolism, decrease apoptosis and support adhesion to titanium healing abutments. In this study, we developed a strontium-loaded alginate hydrogel scaffold which can be easily personalized to fit over any size and shape of implant transmucosal collar or healing abutment. Results indicate that biologically active strontium ions are effectively released from loaded alginate hydrogel material to promote fibroblast viability and migration to repair in vitro wounds similar to that of strontium citrate solution. Overall, this novel strontium-loaded alginate scaffold device displays good biocompatibility and functionality, demonstrating high potential as a system to provide local delivery of strontium to improve peri-implant mucosal healing following implant placement and clinical success.

Near infrared spectroscopic evaluation of biochemical and crimp properties of knee joint ligaments and patellar tendon.

Knee ligaments and tendons play an important role in stabilizing and controlling the motions of the knee. Injuries to the ligaments can lead to abnormal mechanical loading of the other supporting tissues (e.g., cartilage and meniscus) and even osteoarthritis. While the condition of knee ligaments can be examined during arthroscopic repair procedures, the arthroscopic evaluation suffers from subjectivity and poor repeatability. Near infrared spectroscopy (NIRS) is capable of non-destructively quantifying the composition and structure of collagen-rich connective tissues, such as articular cartilage and meniscus. Despite the similarities, NIRS-based evaluation of ligament composition has not been previously attempted. In this study, ligaments and patellar tendon of ten bovine stifle joints were measured with NIRS, followed by chemical and histological reference analysis. The relationship between the reference properties of the tissue and NIR spectra was investigated using partial least squares regression. NIRS was found to be sensitive towards the water (R2CV = .65) and collagen (R2CV = .57) contents, while elastin, proteoglycans, and the internal crimp structure remained undetectable. As collagen largely determines the mechanical response of ligaments, we conclude that NIRS demonstrates potential for quantitative evaluation of knee ligaments.

Dietary Alaska Pollack Protein Induces Acute and Sustainable Skeletal Muscle Hypertrophy in Rats

Our previous studies suggested that Alaska pollack protein (APP) intake increases skeletal muscle mass and that it may cause a slow-to-fast shift in muscle fiber type in rats fed a high-fat diet after 56 days of feeding. In this study, we explored whether dietary APP induces acute and sustainable skeletal muscle hypertrophy in rats fed a normal-fat diet. Male 5-week-old Sprague–Dawley rats were divided into four groups and fed a purified ingredient-based high-fat diet or a purified ingredient-based normal-fat diet with casein or APP, containing the same amount of crude protein. Dietary APP significantly increased gastrocnemius muscle mass (105~110%) after 2, 7 days of feeding, regardless of dietary fat content. Rats were separated into two groups and fed a normal-fat diet with casein or APP. Dietary APP significantly increased gastrocnemius muscle mass (110%) after 56 days of feeding. Dietary APP significantly increased the cross-sectional area of the gastrocnemius skeletal muscle and collagen-rich connective tissue after 7 days of feeding. It decreased the gene expression of Mstn /Myostatin, Trim63/MuRF1, and Fbxo32/atrogin-1, but not other gene expression, such as serum IGF-1 after 7 days of feeding. No differences were observed between casein and APP groups with respect to the percentage of Type I, Type IIA, and Type IIX or IIB fibers, as determined by myosin ATPase staining after 7 days of feeding. In the similar experiment, the puromycin-labeled peptides were not different between dietary casein and APP after 2 days of feeding. These results demonstrate that APP induces acute and sustainable skeletal muscle hypertrophy in rats, regardless of dietary fat content. Dietary APP, as a daily protein source, may be an approach for maintaining or increasing muscle mass.

Is the human sclera a tendon-like tissue? A structural and functional comparison

Collagen rich connective tissues fulfill a variety of important functions throughout the human body, most of which having to resist mechanical challenges. This review aims to compare structural and functional aspects of tendons and sclera, two tissues with distinct location and function, but with striking similarities regarding their cellular content, their extracellular matrix and their low degree of vascularization. The description of these similarities meant to provide potential novel insight for both the fields of orthopedic research and ophthalmology.

Periostin in Periodontics: A Brief Insight

The prime objective of the periodontal treatment is to re-establish the lost periodontal tissue by forming new attachment of periodontal ligament, cementum and the alveolar bone. Periostin is a matricellular protein with its structure closely resembling to that of Drosophila fasciclin. It is expressed in the periodontal ligament, periosteum, alveolar bone, adipose tissue as well as skeletal tissue and has a key role in role in tooth development. This protein molecule has a striking similarity to the molecule beta ig-h3 which is induced by transforming growth factor beta (TGF-beta), promotes the adhesion and spreading of fibroblasts and aids in collagen formation. It is possibly involved in the post surgical regeneration of periodontal hard and soft tissues. Periostin is also linked to dental papilla cells and is associated with hard tissue formation stages of tooth development. This mini review focuses on the various aspects of periostin related to maintenance and development of collagen rich connective tissues around the teeth. Keywords: Periostin, Periodontal ligament, Periodontal regeneration, Alveolar bone.

Anatomical Distribution of Ochronotic Pigment in Alkaptonuric Mice is Associated with Calcified Cartilage Chondrocytes at Osteochondral Interfaces

Alkaptonuria (AKU) is characterised by increased circulating homogentisic acid and deposition of ochronotic pigment in collagen-rich connective tissues (ochronosis), stiffening the tissue. This process over many years leads to a painful and severe osteoarthropathy, particularly affecting the cartilage of the spine and large weight bearing joints. Evidence in human AKU tissue suggests that pigment binds to collagen. The exposed collagen hypothesis suggests that collagen is initially protected from ochronosis, and that ageing and mechanical loading causes loss of protective molecules, allowing pigment binding. Schmorl’s staining has previously demonstrated knee joint ochronosis in AKU mice. This study documents more comprehensively the anatomical distribution of ochronosis in two AKU mouse models (BALB/c Hgd−/−, Hgd tm1a−/−), using Schmorl’s staining. Progression of knee joint pigmentation with age in the two AKU mouse models was comparable. Within the knee, hip, shoulder, elbow and wrist joints, pigmentation was associated with chondrons of calcified cartilage. Pigmented chondrons were identified in calcified endplates of intervertebral discs and the calcified knee joint meniscus, suggesting that calcified tissues are more susceptible to pigmentation. There were significantly more pigmented chondrons in lumbar versus tail intervertebral disc endplates (p = 0.002) and clusters of pigmented chondrons were observed at the insertions of ligaments and tendons. These observations suggest that loading/strain may be associated with increased pigmentation but needs further experimental investigation. The calcified cartilage may be the first joint tissue to acquire matrix damage, most likely to collagen, through normal ageing and physiological loading, as it is the first to become susceptible to pigmentation.

Validity and reliability of measurements of aponeurosis dimensions from magnetic resonance images

Muscle performance is closely related to the structure and function of tendons and aponeuroses, the sheet-like, intramuscular parts of tendons. The architecture of aponeuroses has been difficult to study with magnetic resonance imaging (MRI) because these thin, collagen-rich connective tissues have very short transverse relaxation (T2) times and therefore provide a weak signal with conventional MRI sequences. Here, we validated measurements of aponeurosis dimensions from two MRI sequences commonly used in muscle-tendon research (mDixon and T1-weighted images), and an ultrashort echo time (UTE) sequence designed for imaging tissues with short T2 times. MRI-based measurements of aponeurosis width, length, and area of 20 sheep leg muscles were compared to direct measurements made with three-dimensional (3D) quantitative microdissection. The errors in measurement of aponeurosis width relative to the mean width were 1.8% for UTE, 3.7% for T1, and 18.8% for mDixon. For aponeurosis length, the errors were 7.6% for UTE, 1.9% for T1, and 21.0% for mDixon. Measurements from T1 and UTE scans were unbiased, but mDixon scans systematically underestimated widths, lengths, and areas of the aponeuroses. Using the same methods, we then found high inter-rater reliability (intraclass correlation coefficients >0.92 for all measures) of measurements of the dimensions of the central aponeurosis of the human tibialis anterior muscle from T1-weighted scans. We conclude that valid and reliable measurements of aponeurosis dimensions can be obtained from UTE and from T1-weighted scans. When the goal is to study the macroscopic architecture of aponeuroses, UTE does not hold an advantage over T1-weighted imaging.

Periostin and Human Teeth.

Periostin is a secreted matricellular protein that primarily interacts with type I collagen and fibronectin extracellular matrix proteins, and is widely distributed in tissues rich in collagen-rich connective tissues, including the periodontal ligament. Its expression in these tissues is especially regulated by mechanical load. While the expression and regulation of periostin in the teeth of murine models and cell lines is well known, its presence in human teeth is poorly documented. Here we update and summarize the available data on the distribution of periostin in the human periodontal ligament, gingiva and dental pulp.

Engineering the Second Generation of Therapeutic Cells with Enhanced Targeting of Injured Tissues.

Experimental approaches to improving tissue repair utilize cells and growth factors needed to restore the architecture and function of damaged tissues and organs. Key limitations of these approaches include poor delivery of therapeutic cells and growth factors into injury sites, as well as their short-term retention in target areas. In our earlier studies, we demonstrated that artificial collagen-specific anchor (ACSA) expressed on the surface of therapeutic cells directs them into collagen-rich sites of injury. Moreover, we demonstrated that the ACSA improves the retention of these cells in target sites, thereby promoting tissue repair. To advance the ACSA-based technology, we engineered the second generation of the ACSA-expressing cells able to deliver growth factors to target sites. In this study, we specifically focused on insulin growth factor 1 (IGF1), which enhances the repair of a number of collagen-rich connective tissues, including ligament and tendon. Utilizing gene engineering, we produced IGF1 in the ACSA-expressing cells. Using relevant experimental models, we demonstrated that recombinant IGF1 secreted by these cells maintains its specificity and biological activity. Moreover, our studies show that IGF1 produced by the ACSA-expressing cells cultured in three-dimensional environment promotes the formation of the collagen-rich fibrillar matrix. Furthermore, the engineered cells integrated well with the native collagen-rich tendon tissue. Our study provides strong evidence for the great potential of cells with rationally engineered target-specific receptors to restore damaged connective tissues. Future studies in relevant animal models will determine the utility of these cells in vivo.

Periostin inhibits mechanical stretch-induced apoptosis in osteoblast-like MG-63 cells

Appropriate mechanical stress plays an important role in regulating the proliferation and differentiation of osteoblasts, whereas high-level mechanical stress may be harmful and compromise cell survival. Periostin, a matricellular protein, is essential in maintaining functional integrity of bone and collagen-rich connective tissue in response to mechanical stress. This study investigated whether or not high-level mechanical stretch induces cell apoptosis and the regulatory role of periostin in mechanical stretch-induced apoptosis in osteoblastic cells. Osteoblast-like MG-63 cells were seeded onto Bio-Flex I culture plates and subjected to cyclic mechanical stretching (15% elongation, 0.1Hz) in a Flexercell tension plus system-5000. The same process was applied to cells pre-treated with exogenous human recombinant periostin before mechanical stretching. We used a chromatin condensation and membrane permeability dead cell apoptosis kit to evaluate the stretch-induced cell responses. Expression of caspase-3 and cPARP was examined by immunofluorescent stain and flow cytometry. The expression of periostin in MG-63 cells is involved in the TGF-β signaling pathway. High-level cyclic mechanical stretch induced apoptotic responses in MG-63 osteoblastic cells. The percentages of apoptotic cells and cells expressing cPARP protein increased in the groups of cells subjected to mechanical stretch, but these responses were absent in the presence of exogenous periostin. Our study revealed that high-level mechanical stretch induces apoptotic cell death, and that periostin plays a protective role against mechanical stretch-induced apoptosis in osteoblastic cells.

Effect of age on the occurrence of muscle fiber degeneration associated with myopathies in broiler chickens submitted to feed restriction

To evaluate muscle fiber degeneration (MFD) associated with white striping and wooden breast, pectoralis major of 192 broilers differing for genotype (standard vs. high breast yield), gender, and feeding regime (ad libitum vs. restricted rate 80% from 13 to 21 d of age) were sampled at 14, 21, 28, 35, and 46 d of age for histological analyses by hematoxylin and eosin (H&E) staining to evaluate tissue morphology, Masson's trichrome to identify collagen presence, and Oil red and Nile blue for lipid presence. Microvessels (diameter ≤15 μm), nuclei positive to anti-cleaved lamin A and monoclonal proliferating cell nuclear antigen (PCNA) antisera were counted to assess apoptotic and regenerative processes, respectively. Significant differences were found according to feeding system, age, and their interactions. The frequency of chickens with MFD was higher with ad libitum than restricted feeding (75.0% vs. 62.5%; P = 0.01) and increased with age (18.8%, 28.1%, 75.1%, 96.9%, and 96.9% at 14, 21, 28, 35, and 46 d). However, at 14 d a similar frequency (18.8%) was found in all broilers; at 21 d, MFD occurred more in broilers fed ad libitum than in those under restriction (50.0% vs. 6.3%; P < 0.01); at 28 d differences were reduced (87.5% vs. 62.5%; P = 0.10) to disappear by 35 (100% and 93.8%) and 46 d (96.9% and 96.9%). The number of microvessels decreased with age (20.7 to 9.46; P < 0.001) and the number of nuclei positive to the anti-cleaved lamin A antibody increased. At histology, MFD at 46 d corresponded to loss of typical cross striations, massive necrotic process, degenerating fibers surrounded by inflammatory cells, scattered fibers in an abundant collagen-rich connective tissue, numerous adipose cells; necrotic fibers showed a high percentage of apoptotic nuclei, and regenerating fibers appeared positive to anti-PCNA antibody. In conclusion, MFD soon occurred after 2 wk of growth and increased dramatically within 28 d. Early feed restriction reduced MFD as long as animals were restricted, but no residual effect was recorded after re-alimentation.

Ectopic bone formation by gel-derived bioactive glass-poly-L-lactide-co-glycolide composites in a rabbit muscle model

In this study we aimed to assess the in vivo osteoinductive properties of two composite scaffolds made of PLGA (poly-L-lactide-co-glycolide) and two types of gel-derived bioactive glasses, namely a high silica S2 bioactive glass (S2-PLGA composites) or high lime A2 bioactive glass (A2-PLGA composites). To achieve that, the potential of the composites to induce ectopic bone formation in a rabbit muscle has been examined along with the control PLGA scaffold. Cylinder-like scaffolds of 7  ×  3 mm (width  ×  height) were implanted into pouches created in the latissimus dorsi muscle of 18 New Zealand rabbits. The tissue sections were obtained at 6, 12 or 24 weeks post-surgery (six rabbits per each time point) and stained with hematoxylin-eosin. The process of wound healing, the formation of collagen-rich connective tissue and its transition to cartilage were examined by Sirius red and Alcian blue histological stainings. We also performed immunohistochemical verification of the presence of osteoblast- and osteoclast- like cells in the vicinity of the scaffolds. A typical foreign body reaction and wound healing process was observed for all implanted scaffolds. Osteoblast- and osteoclast-like cells were observed in the vicinity of the scaffolds as determined by the immunohistochemical staining for Osteocalcin, BMP-2 and Cathepsin K. Compared to plain PLGA scaffolds, numerous osteoblast-like cells were observed 12 weeks post implantation near the composites and the scaffolds gradually degraded as bone formation proceeded.S2-PLGA and A2-PLGA composites display osteoinductive properties in vivo. Furthermore, they are more effective at inducing ectopic bone formation in a rabbit muscle compared to plain PLGA. Thus these SBG-PLGA composite scaffolds have potential for clinical applications in dental and/or orthopedic-bone tissue engineering.

Designing dense connective tissues: identifying microenvironmental factors that direct human MSC differentiation in 3D

Event Abstract Back to Event Designing dense connective tissues: identifying microenvironmental factors that direct human MSC differentiation in 3D Jenna Usprech1 and Craig A. Simmons1, 2 1 University of Toronto, Institute of Biomaterials and Biomedical Engineering, Canada 2 University of Toronto, Department of Mechanical and Industrial Engineering, Canada Introduction: Microenvironmental factors such as biomaterial physical and biochemical properties, external mechanical stimuli, and soluble chemical cues integrate in vivo to direct the differentiation of mesenchymal stromal cells (MSCs). Identifying what factor combinations promote the differentiation of MSCs into myofibroblasts (the tissue producing cells of developing valves, ligaments, skin, tendons and periodontal ligaments) is a significant challenge when designing for dense, type I collagen-rich connective tissues. To date, select combinations of microenvironmental factors have been studied in 2D, but systematic consideration of the integration of factors in 3D culture has not been explored fully. Materials and Methods: We developed a 3D screening platform to systematically study MSC responses to microenvironmental stimuli. Permutations of the cell adhesion sequences RGD, DGEA and YIGSR at different concentrations (0-2 mM) were incorporated into 5-11 wt% (5-22 kPa compressive moduli) polyethylene glycol norbornene (PEG-NB) hydrogel arrays. Since PEG-NB is inert and easily crosslinked with UV light, adhesion ligands can be tuned independently from biomaterial properties, enabling us to identify the contribution of individual factors in a combinatorial fashion. Human bone marrow-derived MSCs were encapsulated and cultured in the 3D arrays for 7 days with 0 or 5 ng/mL TGF-β1. The extent of α-smooth muscle actin (αSMA) expression and collagen type I deposition were modeled using least squares estimation and regression on 3D confocal data (Figure 1A) that was analysed using Imaris (Figure 1B). Each experiment was replicated four times and statistical analyses were performed in JMP on the pooled data. Results and Discussion: Regression analyses of the screening data revealed that in the presence of TGF-β1, PEG-NB hydrogel wt% was the most significant factor influencing myofibroblast differentiation (p < 0.0001), with greater αSMA staining intensity manifesting at lower gel wt%. These data are in contrast to MSC myofibroblastic differentiation in 2D, which is promoted by stiffer substrates. With TGF-β1, RGD concentration was correlated with αSMA staining intensity in a positive, biphasic manner and demonstrated a significant, non-linear dependence with PEG-NB wt% (p = 0.02) (Figure 2A). Interestingly, αSMA staining intensity could only be predictably modeled by regression in cultures that were supplemented with TGF-β1. These observations suggest that while MSCs can express αSMA in the absence of exogenous TGF-β1, αSMA expression is only sensitive to material properties in the presence of TGF-β1. Collagen deposition was predominantly negatively correlated with PEG-NB hydrogel wt% (p<0.0001) with or without TGF-β1, with slight non-linear dependence between RGD and DGEA (p = 0.01) (Figure 2B) or linear dependence with RGD (p = 0.04) in the absence or presence of TGF-β1, respectively. Conclusion: The newly developed 3D screening platform enables the systematic identification of optimal conditions for connective tissue engineering when considering multiple interacting microenvironmental factors. The data presented here demonstrate that conditions which promote a myofibroblast phenotype in 2D do not translate completely to the more complex and integrative 3D environment. Keywords: Cell Differentiation, screening, 3D scaffold, instructive microenvironment Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: New Frontier Oral Topic: Biomaterials in constructing tissue substitutes Citation: Usprech J and Simmons CA (2016). Designing dense connective tissues: identifying microenvironmental factors that direct human MSC differentiation in 3D. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.02714 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Jenna Usprech Craig A Simmons Google Jenna Usprech Craig A Simmons Google Scholar Jenna Usprech Craig A Simmons PubMed Jenna Usprech Craig A Simmons Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.