Tuning Physical Properties of GelMA Hydrogels through Microarchitecture for Engineering Osteoid Tissue.

Gelatin methacryloyl (GelMA) hydrogels have gained significant attention due to their biocompatibility and tunable properties. Here, a new approach to engineer GelMA-based matrices to mimic the osteoid matrix is provided. Two cross-linking methods were employed to mimic the tissue stiffness: standard cross-linking (SC) based on visible light exposure (VL) and dual cross-linking (DC) involving physical gelation, followed by VL. It was demonstrated that by reducing the GelMA concentration from 10% (G10) to 5% (G5), the dual-cross-linked G5 achieved a compressive modulus of ∼17 kPa and showed the ability to support bone formation, as evidenced by alkaline phosphatase detection over 3 weeks of incubation in osteogenic medium. Moreover, incorporating poly(ethylene) oxide (PEO) into the G5 and G10 samples was found to hinder the fabrication of highly porous hydrogels, leading to compromised cell survival and reduced osteogenic differentiation, as a consequence of incomplete PEO removal.

A vertical additive-lathe printing system for the fabrication of tubular constructs using gelatin methacryloyl hydrogel.

Reproducing both the mechanical and biological performance of native blood vessels remains an ongoing challenge in vascular tissue engineering. Additive-lathe printing offers an attractive method of fabricating long tubular constructs as a potential vascular graft for the treatment of cardiovascular diseases. Printing hydrogels onto rotating horizontal mandrels often leads to sagging, resulting in poor and variable mechanical properties. In this study, an additive-lathe printing system with a vertical mandrel to fabricate tubular constructs is presented. Various concentrations of gelatin methacryloyl (gelMA) hydrogel were used to print grafts on the rotating mandrel in a helical pattern. The printing parameters were selected to achieve the bonding of consecutive gelMA filaments to improve the quality of the printed graft. The hydrogel filaments were fused properly under the action of gravity on the vertical mandrel. Thus, the vertical additive-lathe printing system was used to print uniform wall thickness grafts, eliminating the hydrogel sagging problem. Tensile testing performed in both circumferential and longitudinal direction revealed that the anisotropic properties of printed gelMA constructs were similar to those observed in the native blood vessels. In addition, no leakage was detected through the walls of the gelMA grafts during burst pressure measurement. Therefore, the current printing setup could be utilized to print vascular grafts for the treatment of cardiovascular diseases.

Engineering three-dimensional bone macro-tissues by guided fusion of cell spheroids.

Introduction: Bioassembly techniques for the application of scaffold-free tissue engineering approaches have evolved in recent years toward producing larger tissue equivalents that structurally and functionally mimic native tissues. This study aims to upscale a 3-dimensional bone in-vitro model through bioassembly of differentiated rat osteoblast (dROb) spheroids with the potential to develop and mature into a bone macrotissue. Methods: dROb spheroids in control and mineralization media at different seeding densities (1 × 104, 5 × 104, and 1 × 105 cells) were assessed for cell proliferation and viability by trypan blue staining, for necrotic core by hematoxylin and eosin staining, and for extracellular calcium by Alizarin red and Von Kossa staining. Then, a novel approach was developed to bioassemble dROb spheroids in pillar array supports using a customized bioassembly system. Pillar array supports were custom-designed and printed using Formlabs Clear Resin® by Formlabs Form2 printer. These supports were used as temporary frameworks for spheroid bioassembly until fusion occurred. Supports were then removed to allow scaffold-free growth and maturation of fused spheroids. Morphological and molecular analyses were performed to understand their structural and functional aspects. Results: Spheroids of all seeding densities proliferated till day 14, and mineralization began with the cessation of proliferation. Necrotic core size increased over time with increased spheroid size. After the bioassembly of spheroids, the morphological assessment revealed the fusion of spheroids over time into a single macrotissue of more than 2.5 mm in size with mineral formation. Molecular assessment at different time points revealed osteogenic maturation based on the presence of osteocalcin, downregulation of Runx2 (p < 0.001), and upregulated alkaline phosphatase (p < 0.01). Discussion: With the novel bioassembly approach used here, 3D bone macrotissues were successfully fabricated which mimicked physiological osteogenesis both morphologically and molecularly. This biofabrication approach has potential applications in bone tissue engineering, contributing to research related to osteoporosis and other recurrent bone ailments.

Quantity of the antigens of Streptococcus mutans serotype e and Candida albicans and its correlation with the salivary flow rate in early childhood caries.

Background: Streptococcus mutans involved in caries pathogenesis is classified into four serotypes, namely serotypes c, e, f, and k. Candida albicans can be found in the plaque of children with early childhood caries (ECC). Aims: The aim of this study was to analyze the quantity of the antigens of S. mutans serotype e and C. albicans and its correlation with the salivary flow rate in ECC. Materials and Methods: The antigen quantities of caries plaque samples and caries-free were determined using an enzyme-linked immunoassay with 450-nm optical density. Results: There was a significant difference between the quantity of S. mutans serotype e and C. albicans antigens in each salivary flow rate category (P < 0.05). The relationship between the antigen quantity of S. mutans serotype e and C. albicans was r = 0.624 (P > 0.05) for caries plaque samples and r = 0.628 (P > 0.05) for caries-free samples. Conclusion: the antigen quantities of S. mutans serotype e and C. albicans and the salivary flow rate might correlate to the pathogenesis of ECC.

Serious complications and recurrences after pelvic organ prolapse surgery for 2309 women in the VIGI-MESH registry.

OBJECTIVE: To assess the incidence of serious complications and reoperations for recurrence after surgery for pelvic organ prolapse (POP) and compare the three most common types of repair. DESIGN: Prospective cohort study using a registry. SETTING: Nineteen French surgical centres. POPULATION: A total of 2309 women participated between 2017 and 2019. METHODS: A multivariate analysis including an inverse probability of treatment weighting approach was used to obtain three comparable groups. MAIN OUTCOME MEASURES: Serious complications and subsequent reoperations for POP recurrence. RESULTS: The median follow-up time was 17.6 months. Surgeries were native tissue vaginal repairs (n = 504), transvaginal mesh placements (n = 692) and laparoscopic sacropexies with mesh (n = 1113). Serious complications occurred among 52 women (2.3%), and reoperation for POP recurrence was required for 32 women (1.4%). At 1 year the cumulative weighted incidence of serious complications was 1.8% for native tissue vaginal repair, 3.9% for transvaginal mesh and 2.2% for sacropexy, and the rates for reoperation for recurrence of POP were 1.5, 0.7 and 1.1%, respectively. Compared with native tissue vaginal repair, the risk of serious complications was higher in the transvaginal mesh group (weighted hazard ratio, wHR 3.84, 95% CI 2.43-6.08) and the sacropexy group (wHR 2.48, 95% CI 1.45-4.23), whereas the risk of reoperation for prolapse recurrence was lower in both the transvaginal mesh (wHR 0.22, 95% CI 0.13-0.39) and sacropexy (wHR 0.29, 95% CI 0.18-0.47) groups. CONCLUSIONS: Our results suggest that native tissue vaginal repairs have the lowest risk of serious complications but the highest risk of reoperation for recurrence. These results are useful for informing women and for shared decision making. TWEETABLE ABSTRACT: Laparoscopic sacropexy had fewer serious complications than transvaginal mesh and fewer reoperations for recurrence than vaginal repair.

Long-term outcomes of primary cystocele repair by transvaginal mesh surgery versus laparoscopic mesh sacropexy: extended follow up of the PROSPERE multicentre randomised trial.

OBJECTIVE: To compare the effectiveness and safety of laparoscopic sacropexy (LS) and transvaginal mesh (TVM) at 4 years. DESIGN: Extended follow up of a randomised trial. SETTING: Eleven centres. POPULATION: Women with cystocele stage ≥2 (pelvic organ prolapse quantification [POP-Q], aged 45-75 years without previous prolapse surgery. METHODS: Synthetic non-absorbable mesh placed in the vesicovaginal space and sutured to the promontory (LS) or maintained by arms through pelvic ligaments and/or muscles (TVM). MAIN OUTCOME MEASURES: Functional outcomes (pelvic floor distress inventory [PFDI-20] as primary outcome); anatomical assessment (POP-Q), composite outcome of success; re-interventions for complications. RESULTS: A total of 220 out of 262 randomised patients have been followed at 4 years. PFDI-20 significantly improved in both groups and was better (but below the minimal clinically important difference) after LS (mean difference -7.2 points; 95% CI -14.0 to -0.05; P = 0.029). The improvement in quality of life and the success rate (LS 70%, 61-81% versus TVM 71%, 62-81%; hazard ratio 0.92, 95% CI 0.55-1.54; P = 0.75) were similar. POP-Q measurements did not differ, except for point C (LS -57 mm versus TVM -48 mm, P = 0.0093). The grade III or higher complication rate was lower after LS (2%, 0-4.7%) than after TVM (8.7%, 3.4-13.7%; hazard ratio 4.6, 95% CI 1.007-21.0, P = 0.049)). CONCLUSIONS: Both techniques provided improvement and similar success rates. LS had a better benefit-harm balance with fewer re-interventions due to complications. TVM remains an option when LS is not feasible. TWEETABLE ABSTRACT: At 4 years, Laparoscopic Sacropexy (LS) had a better benefit-harm balance with fewer re-interventions due to complications than Trans-Vaginal Mesh (TVM).

Elastic Bioresorbable Polymeric Capsules for Osmosis-Driven Delayed Burst Delivery of Vaccines.

Single-administration vaccine delivery systems are intended to improve the efficiency and efficacy of immunisation programs in both human and veterinary medicine. In this work, an osmotically triggered delayed delivery device was developed that was able to release a payload after a delay of approximately 21 days, in a consistent and reproducible manner. The device was constructed out of a flexible poly(ε-caprolactone) photo-cured network fabricated into a hollow tubular shape, which expelled approximately 10% of its total payload within 2 days after bursting. Characterisation of the factors that control the delay of release demonstrated that it was advantageous to adjust material permeability and device wall thickness over manipulation of the osmogent concentration in order to maintain reproducibility in burst delay times. The photo-cured poly(ε-caprolactone) network was shown to be fully degradable in vitro, and there was no evidence of cytotoxicity after 11 days of direct contact with primary dermal fibroblasts. This study provides strong evidence to support further development of flexible biomaterials with the aim of continuing improvement of the device burst characteristics in order to provide the greatest chance of the devices succeeding with in vivo vaccine booster delivery.

Diabetes care and pregnancy outcomes for women with pregestational diabetes in Ireland.

AIMS: Pre-gestational diabetes mellitus (PGDM) is associated with adverse outcomes. We aimed to examine pregnancies affected by PGDM; report on these pregnancy outcomes and compare outcomes for patients with type 1 versus type 2 diabetes mellitus; compare our findings to published Irish and United Kingdom (UK) data and identify potential areas for improvement. METHODS: Between 2016 and 2018 information on 679 pregnancies from 415 women with type 1 Diabetes Mellitus and 244 women with type 2 diabetes was analysed. Data was collected on maternal characteristics; pregnancy preparation; glycaemic control; pregnancy related complications; foetal and maternal outcomes; unscheduled hospitalisations; congenital anomalies and perinatal deaths. RESULTS: Only 15.9% of women were adequately prepared for pregnancy. Significant deficits were identified in availability and attendance at pre-pregnancy clinic, use of folic acid, attaining appropriate glycaemic targets and appropriate retinal screening. The majority of pregnancies (n = 567, 83.5%) resulted in a live birth but the large number of infants born large for gestational age (LGA) (n = 280, 49.4%), born prematurely <37 weeks and requiring neonatal intensive care unit (NICU) admission continue to be significant issues. CONCLUSIONS: This retrospective cohort study identifies multiple targets for improvements in the provision of care to women with pre-gestational DM which are likely to translate into better pregnancy outcomes.

Emulating Human Tissues and Organs: A Bioprinting Perspective Toward Personalized Medicine.

The lack of in vitro tissue and organ models capable of mimicking human physiology severely hinders the development and clinical translation of therapies and drugs with higher in vivo efficacy. Bioprinting allow us to fill this gap and generate 3D tissue analogues with complex functional and structural organization through the precise spatial positioning of multiple materials and cells. In this review, we report the latest developments in terms of bioprinting technologies for the manufacturing of cellular constructs with particular emphasis on material extrusion, jetting, and vat photopolymerization. We then describe the different base polymers employed in the formulation of bioinks for bioprinting and examine the strategies used to tailor their properties according to both processability and tissue maturation requirements. By relating function to organization in human development, we examine the potential of pluripotent stem cells in the context of bioprinting toward a new generation of tissue models for personalized medicine. We also highlight the most relevant attempts to engineer artificial models for the study of human organogenesis, disease, and drug screening. Finally, we discuss the most pressing challenges, opportunities, and future prospects in the field of bioprinting for tissue engineering (TE) and regenerative medicine (RM).

Prolonged recovery of 3D printed, photo-cured polylactide shape memory polymer networks.

Shape memory polymers are materials that are able to retain a deformed state until an external stimulus, most typically heat, triggers recovery to the original geometry. Whereas typically, shape memory polymers are required to recover fast (seconds to minutes), many applications, particularly in the medical field, would benefit from a slow recovery (days to weeks). In this work, we exploit the broad glass transition range of photo-cured poly(D,L-lactide) dimethacrylate networks to obtain recovery times of up to 2 weeks, at 11 °C below the peak glass transition temperature of 58 °C. Recovery times decreased considerably for higher recovery temperatures, down to ∼10 min at 55 °C. A large spread in glass transition values (53.3-61.0 °C) was observed between samples, indicating poor reproducibility in sample preparation and, hence, in predicting shape recovery kinetics for individual samples. Furthermore, a staged recovery was observed with different parts of the samples recovering at different times. The ability to prepare complex structures using digital light processing stereolithography 3D printing from these polymers was confirmed. To the best of our knowledge, this work provides the first experimental evidence of prolonged recovery of shape memory polymers.

3D Printing in Suspension Baths: Keeping the Promises of Bioprinting Afloat.

Extrusion-based 3D printers have been adopted in pursuit of engineering functional tissues through 3D bioprinting. However, we are still a long way from the promise of fabricating constructs approaching the complexity and function of native tissues. A major challenge is presented by the competing requirements of biomimicry and manufacturability. This opinion article discusses 3D printing in suspension baths as a novel strategy capable of disrupting the current bioprinting landscape. Suspension baths provide a semisolid medium to print into, voiding many of the inherent flaws of printing onto a flat surface in air. We review the state-of-the-art of this approach and extrapolate toward future possibilities that this technology might bring, including the fabrication of vascularized tissue constructs.

An Irish National Diabetes in Pregnancy Audit: aiming for best outcomes for women with diabetes.

AIMS: The purpose of this study was to identify the number of pregnancies affected by pre-gestational diabetes in the Republic of Ireland; to report on pregnancy outcomes and to identify areas for improvement in care delivery and clinical outcomes. METHODS: Healthcare professionals caring for women with pre-gestational diabetes during pregnancy were invited to participate in this retrospective study. Data pertaining to 185 pregnancies in women attending 15 antenatal centres nationally were collected and analysed. Included pregnancies had an estimated date of delivery between 1 January and 31 December 2015. RESULTS: The cohort consisted of 122 (65.9%) women with Type 1 diabetes and 56 (30.3%) women with Type 2 diabetes. The remaining 7 (3.8%) pregnancies were to women with maturity-onset diabetes of the young (MODY) (n = 6) and post-transplant diabetes (n = 1). Overall women were poorly prepared for pregnancy and lapses in specific areas of service delivery including pre-pregnancy care and retinal screening were identified. The majority of pregnancies 156 (84.3%) resulted in a live birth. A total of 103 (65.5%) women had a caesarean delivery and 58 (36.9%) infants were large for gestational age. CONCLUSIONS: This audit identifies clear areas for improvement in delivery of care for women with diabetes in the Republic of Ireland before and during pregnancy.

Complications after pelvic floor repair surgery (with and without mesh): short-term incidence after 1873 inclusions in the French VIGI-MESH registry.

OBJECTIVE: To assess the short-term incidence of serious complications of surgery for urinary incontinence or pelvic organ prolapse. DESIGN: Prospective longitudinal cohort study using a surgical registry. SETTING: Thirteen public hospitals in France. POPULATION: A cohort of 1873 women undergoing surgery between February 2017 and August 2018. METHODS: Preliminary analysis of serious complications after a mean follow-up of 7 months (0-18 months), according to type of surgery. Surgeons reported procedures and complications, which were verified by the hospitals' information systems. MAIN OUTCOME MEASURES: Serious complication requiring discontinuation of the procedure or subsequent surgical intervention, life-threatening complication requiring resuscitation, or death. RESULTS: Fifty-two women (2.8%, 95% CI 2.1-3.6%) experienced a serious complication either during surgery, requiring the discontinuation of the procedure, or during the first months of follow-up, necessitating a subsequent reoperation. One woman also required resuscitation; no women died. Of 811 midurethral slings (MUSs), 11 were removed in part or totally (1.4%, 0.7-2.3%), as were two of 391 transvaginal meshes (0.5%, 0.1-1.6%), and four of 611 laparoscopically placed mesh implants (0.7%, 0.2-1.5%). The incidence of serious complications 6 months after the surgical procedure was estimated to be around 3.5% (2.0-5.0%) after MUS alone, 7.0% (2.8-11.3%) after MUS with prolapse surgery, 1.7% (0.0-3.8%) after vaginal native tissue repair, 2.8% (0.9-4.6%) after transvaginal mesh, and 1.0% (0.1-1.9%) after laparoscopy with mesh. CONCLUSIONS: Early serious complications are relatively rare. Monitoring must be continued and expanded to assess the long-term risk associated with mesh use and to identify its risk factors. TWEETABLE ABSTRACT: Short-term serious complications are rare after surgery for urinary incontinence or pelvic organ prolapse, even with mesh.

Integrated Care Models for Managing and Preventing Frailty: A Systematic Review for the European Joint Action on Frailty Prevention (ADVANTAGE JA).

Frailty requires concerted integrated approaches to prevent functional decline. Although there is evidence that integrating care is effective for older people, there is insufficient data on outcomes from studies implementing integrated care to prevent and manage frailty. We systematically searched PubMed and Cochrane Library database for peer-reviewed medical literature on models of care for frailty, published from 2002 to 2017. We considered the effective and transferable components of the models of care and evidence of economic impact, where available. Information on European Union-funded projects or those registered with the European Innovation Partnership on Active and Healthy Ageing, and grey literature (including good practices) were also considered. We found 1,065 potential citations and 170 relevant abstracts. After excluding reports on specific diseases, processes or interventions and service models that did not report data, 42 full papers met the inclusion criteria. The evidence showed that few models of integrated care were specifically designed to prevent and tackle frailty in the community and at the interface between primary and secondary (hospital) care. Current evidence supports the case for a more holistic and salutogenic response to frailty, blending a chronic care approach with education, enablement and rehabilitation to optimise function, particularly at times of a sudden deterioration in health, or when transitioning between home, hospital or care home. In all care settings, these approaches should be supported by comprehensive assessment and multidimensional interventions tailored to modifiable physical, psychological, cognitive and social factors.

[Applications of 3D printing in medicine; 5 years later]. / Mogelijkheden van 3D-printen in de geneeskunde.

5 years ago, we described the emergence of 3D printing in medicine. It was about 3D printing of anatomical structures, patient-specific drilling guides, cutting templates and implants and printing of living cells, growth factors and biomaterials ('bioprinting'). Surgeons are increasingly making use of 3D printing possibilities in preparation of surgeries on patients with complicated anatomies. Using tangible 3D models, it is easier for surgeons to prepare for surgeries and discussions with patients. They can also use 3D models as a tool to help with the training of young surgeons. Permanent titanium implants are increasingly being printed. Bioprinting is still in its infancy and there are no direct clinical applications yet. As we already predicted 5 years ago, many hurdles still have to be taken before broad clinical application of bioprinted products will become a reality.

Visible Light Cross-Linking of Gelatin Hydrogels Offers an Enhanced Cell Microenvironment with Improved Light Penetration Depth.

In this study, the cyto-compatibility and cellular functionality of cell-laden gelatin-methacryloyl (Gel-MA) hydrogels fabricated using a set of photo-initiators which absorb in 400-450 nm of the visible light range are investigated. Gel-MA hydrogels cross-linked using ruthenium (Ru) and sodium persulfate (SPS), are characterized to have comparable physico-mechanical properties as Gel-MA gels photo-polymerized using more conventionally adopted photo-initiators, such as 1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propan-1-one (Irgacure 2959) and lithium phenyl(2,4,6-trimethylbenzoyl) phosphinate (LAP). It is demonstrated that the Ru/SPS system has a less adverse effect on the viability and metabolic activity of human articular chondrocytes encapsulated in Gel-MA hydrogels for up to 35 days. Furthermore, cell-laden constructs cross-linked using the Ru/SPS system have significantly higher glycosaminoglycan content and re-differentiation capacity as compared to cells encapsulated using I2959 and LAP. Moreover, the Ru/SPS system offers significantly greater light penetration depth as compared to the I2959 system, allowing thick (10 mm) Gel-MA hydrogels to be fabricated with homogenous cross-linking density throughout the construct. These results demonstrate the considerable advantages of the Ru/SPS system over traditional UV polymerizing systems in terms of clinical relevance and practicability for applications such as cell encapsulation, biofabrication, and in situ cross-linking of injectable hydrogels.

Bio-resin for high resolution lithography-based biofabrication of complex cell-laden constructs.

Lithography-based three-dimensional (3D) printing technologies allow high spatial resolution that exceeds that of typical extrusion-based bioprinting approaches, allowing to better mimic the complex architecture of biological tissues. Additionally, lithographic printing via digital light processing (DLP) enables fabrication of free-form lattice and patterned structures which cannot be easily produced with other 3D printing approaches. While significant progress has been dedicated to the development of cell-laden bioinks for extrusion-based bioprinting, less attention has been directed towards the development of cyto-compatible bio-resins and their application in lithography-based biofabrication, limiting the advancement of this promising technology. In this study, we developed a new bio-resin based on methacrylated poly(vinyl alcohol) (PVA-MA), gelatin-methacryloyl (Gel-MA) and a transition metal-based visible light photoinitiator. The utilization of a visible light photo-initiating system displaying high molar absorptivity allowed the bioprinting of constructs with high resolution features, in the range of 25-50 µm. Biofunctionalization of the resin with 1 wt% Gel-MA allowed long term survival (>90%) of encapsulated cells up to 21 d, and enabled attachment and spreading of endothelial cells seeded on the printed hydrogels. Cell-laden hydrogel constructs of high resolution with complex and ordered architecture were successfully bioprinted, where the encapsulated cells remained viable, homogenously distributed and functional. Bone and cartilage tissue synthesis was confirmed by encapsulated stem cells, underlining the potential of these DLP-bioprinted hydrogels for tissue engineering and biofabrication. Overall, the PVA-MA/Gel-MA bio-resin is a promising material for biofabrication and provides important cues for the further development of lithography-based bioprinting of complex, free-form living tissue analogues.

Comparing Hydrogels for Human Nucleus Pulposus Regeneration: Role of Osmolarity During Expansion.

Hydrogels can facilitate nucleus pulposus (NP) regeneration, either for clinical application or research into mechanisms of regeneration. However, many different hydrogels and culture conditions for human degenerated NP have been employed, making literature data difficult to compare. Therefore, we compared six different hydrogels of natural polymers and investigated the role of serum in the medium and of osmolarity during expansion or redifferentiation in an attempt to provide comparators for future studies. Human NP cells of Thompson grade III discs were cultured in alginate, agarose, fibrin, type II collagen, gelatin methacryloyl (gelMA), and hyaluronic acid-poly(ethylene glycol) hydrogels. Medium containing fetal bovine serum and a serum-free (SF) medium were compared in agarose, gelMA, and type II collagen hydrogels. Isolation and expansion of NP cells in low compared to high osmolarity medium were performed before culture in agarose and type II collagen hydrogels in media of varying osmolarity. NP cells in agarose produced the highest amounts of proteoglycans, followed by cells in type II collagen hydrogels. The absence of serum reduced the total amount of proteoglycans produced by the cells, although incorporation efficiency was higher in type II collagen hydrogels in the absence than in the presence of serum. Isolation and expansion of NP cells in high osmolarity medium improved proteoglycan production during culture in hydrogels, but variation in osmolarity during redifferentiation did not have any effect. Agarose hydrogels seem to be the best option for in vitro culture of human NP cells, but for clinical application, type II collagen hydrogels may be better because, as opposed to agarose, it degrades in time. Although culture in SF medium reduces the amount of proteoglycans produced during redifferentiation culture, isolating and expanding the cells in high osmolarity medium can largely compensate for this loss.

Mutans Streptococci counts from saliva and its protein profile in early childhood caries.

AIM: This study aims to analyze the number Mutans Streptococci (MS) and its protein profile from the saliva of early childhood caries (ECC) and caries-free subjects. METHODS: MS counts were cultured from saliva samples, and the protein profile of MS was determined from ECC and caries-free subjects. The number of colonies were counted, and the protein bands with the molecular weight of 13, 29, 39, 41.3, 74, and 95 kDa were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis method. RESULTS: We found that the number of colonies from saliva of ECC patients was higher than those caries-free (22.20 × 106 CFU/ml vs. 19.16 × 106 CFU/ml, p < 0.05). There are higher expression frequencies in protein 29, 39, 41.3, and 74 kDa of MS in ECC than caries-free subjects. CONCLUSIONS: There is the higher number of MS colonies and difference of MS protein profile isolated from saliva among children with ECC and caries-free counterparts.

Focal adhesion signaling affects regeneration by human nucleus pulposus cells in collagen- but not carbohydrate-based hydrogels.

Hydrogel-based 3D cell cultures are an emerging strategy for the regeneration of cartilage. In an attempt to regenerate dysfunctional intervertebral discs, nucleus pulposus (NP) cells can be cultured in hydrogels of various kinds and physical properties. Stiffness sensing through focal adhesions is believed to direct chondrogenesis, but the mechanisms by which this works are largely unknown. In this study we compared focal adhesion formation and glycosaminoglycan (GAG) deposition by NP cells in a range of hydrogels. Using a focal adhesion kinase (FAK) inhibitor, we demonstrated that focal adhesion signaling is involved in the response of NP cells in hydrogels that contain integrin binding sites (i.e. methacrylated gelatin (gelMA) and type II collagen), but not in hydrogels deplete from integrin binding sites such as alginate and agarose, or CD44-binding hydrogels based on hyaluronic acid. As a result of FAK inhibition we observedenhanced proteoglycan production in gelMA, but decreased production in type II collagen hydrogels, which could be explained by alteration in cell fate as supported by the increase in the adipogenic marker peroxisome proliferator-activated receptor gamma (PPARy). Furthermore, GAG deposition was inversely proportional to polymer concentration in integrin-binding gelMA, while no direct relationship was found for the non-integrin binding gels alginate and agarose. This corroborates our finding that focal adhesion formation plays an important role in NP cell response to its surrounding matrix. STATEMENT OF SIGNIFICANCE: Biomaterials are increasingly being investigated for regenerative medicine applications, including regeneration of the nucleus pulposus. Cells interact with their environment and are influenced by extracellular matrix or polymer properties. Insight in these interactions can improve regeneration and helps to understand degeneration processes. The role of focal adhesion formation in the regenerative response of nucleus pulposus cells is largely unknown. Therefore, the relation between materials, stiffness and focal adhesion formation is studied here.