ACCEPTED MANUSCRIPT 1 Substrate stiffness and contractile behaviour modulate the functional maturation of osteoblasts on a collagen GAG scaffold

Please cite this article as: Keogh, M.B., Brien, F.J., Daly, J.S., Substrate stiffness and contractile behaviour modulate the functional maturation of osteoblasts on a collagen GAG scaffold, Acta Biomaterialia (2010), doi: 10.1016/ j.actbio. 2010.06.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. showed that all CG substrates allowed for cellular attachment, infiltration and osteogenic differentiation. ACCEPTED MANUSCRIPT CG scaffolds treated with EDAC and GLUT, were mechanically stiffer, retained their original scaffold structure and resisted cellular contraction. Consequently they facilitated a 2-fold greater cell number probably due to pore architecture being maintained allowing for improved diffusion of nutrients. On the other hand, the less stiff substrates crosslinked with DHT allowed for increased cell-mediated scaffold contraction; contracting by 70% following 6 weeks (p<0.01) of culture. This reduction in scaffold area resulted in cells reaching the centre of the scaffold quicker up to 4 weeks; however, at 6 weeks all scaffolds showed similar levels of cellular infiltration with higher cell numbers found on the stiffer EDAC and GLUT-treated scaffolds. Analysis of osteogenesis showed, that scaffolds crosslinked with DHT expressed higher levels of the late stage bone formation markers osteopontin and osteocalcin (p<0.01) and increased levels of mineralisation. In conclusion, the more compliant CG scaffolds allowed for cellmediated contraction and supported a greater level osteogenic maturation of MC3T3 cells while the stiffer, non contractible scaffolds resulted in lower levels of cell maturation but higher cell numbers on the scaffold. Therefore, we find scaffold stiffness has different effects on differentiation and cell number whereby the increased cell-mediated contraction facilitated by the less stiff scaffolds positively modulates osteoblast differentiation while reducing cell numbers

3 hours of perfusion culture prior to 28 days of static culture, enhances osteogenesis by human cells in a collagen GAG scaffold.

In tissue engineering bioreactors can be used to aid in the in vitro development of new tissue by providing biochemical and physical regulatory signals to cells and encouraging them to undergo differentiation and/or to produce extracellular matrix prior to in vivo implantation. This study examined the effect of short term flow perfusion bioreactor culture, prior to long term static culture, on human osteoblast cell distribution and osteogenesis within a collagen glycosaminoglycan (CG) scaffold for bone tissue engineering. Human Foetal Osteoblasts (hFOB 1.19) were seeded onto CG scaffolds and pre-cultured for 6 days. Constructs were then placed into the bioreactor and exposed to 3×1hr bouts of steady flow (1ml/min) separated by 7hrs of no flow over a 24hr period. The constructs were then cultured under static osteogenic conditions for up to 28 days. Results show that the bioreactor and static culture control groups displayed similar cell numbers and metabolic activity. Histologically however, peripheral cell-encapsulation was observed in the static controls, whereas, improved migration and homogenous cell distribution was seen in the bioreactor groups. Gene expression analysis showed that all osteogenic markers investigated displayed greater levels of expression in the bioreactor groups compared to static controls. While static groups showed increased mineral deposition; mechanical testing revealed that there was no difference in the compressive modulus between bioreactor and static groups. In conclusion, a flow perfusion bioreactor improved construct homogeneity by preventing peripheral encapsulation whilst also providing an enhanced osteogenic phenotype over static controls. © 2010 Wiley Periodicals, Inc

Improved contractile potential in detrusor microtissues from pediatric patients with end stage lower urinary tract dysfunction

Autologous cell-based tissue engineering has been proposed as a treatment option for end stage lower urinary tract dysfunction (ESLUTD). However, it is generally accepted that cells isolated from patient bladders retain the pathological properties of their tissue of origin and therefore need to be improved before they can serve as a cell source for tissue engineering applications. We hypothesize that human three-dimensional (3D) microtissues of detrusor smooth muscle cells (SMCs) are valuable ex vivo disease models and potent building blocks for bladder tissue engineering. Detrusor SMCs isolated from bladder wall biopsies of pediatric ESLUTD patients and healthy controls were expanded and cultured into 3D microtissues. Gene and protein analyses were performed to explore the effect of microtissue formation on SMC viability, contractile potential, bladder wall specific extracellular matrix (ECM) composition and mediators of ECM remodeling. Through microtissue formation, remodeling and intensified cell-cell interactions, the ESLUTD SMCs lost their characteristic disease phenotype. These microtissues exhibited similar patterns of smooth muscle related contractile proteins and essential bladder wall-specific ECM components as microtissues from healthy control subjects. Thus, the presented data suggest improved contractile potential and ECM composition in detrusor SMC microtissues from pediatric ESLUTD patients. These findings are of great relevance, as 3D detrusor SMC microtissues might be an appropriate cell source for autologous cell-based bladder tissue engineering

Mechanomodulatory biomaterials prospects in scar prevention and treatment

Scarring is a major clinical issue that affects a considerable number of patients. The associated problems go beyond the loss of skin functionality, as scars bring aesthetic, psychological, and social difficulties. Therefore, new strategies are required to improve the process of healing and minimize scar formation. Research has highlighted the important role of mechanical forces in the process of skin tissue repair and scar formation, in addition to the chemical signalling. A more complete understanding of how engi- neered biomaterials can modulate these mechanical stimuli and modify the mechanotransduction signals in the wound microenvironment is expected to enable scar tissue reduction. The present review aims to provide an overview of our current understanding of skin biomechanics and mechanobiology underlying wound healing and scar formation, with an emphasis on the development of novel mechanomodulatory wound dressings with the capacity to offload mechanical tension in the wound environment. Further- more, a broad overview of current challenges and future perspectives of promising mechanomodulatory biomaterials for this application are provided.The authors would like to acknowledge Portuguese Foun dation for Science and Technology (FCT) for funding the research project Dressing4Scars M-ERA-NET2/0013/2016, and LP da Silva (2020.01541.CEECIND/CP1600/CT0024), and to Norte-01-0145-FEDER-02219015 (MT Cerqueira)

The effect of biologicaly active substances on the structure and properties of collagenous substrates

Diplomová práce se zabývá přípravou 3D porézních kolagenových skafoldů metodou lyofilizace a jejich modifikací bioaktivními látkami. K modifikaci byly použity přírodní polysacharidy – chitosan, vápenatá oxidovaná celulóza a chitin/chitosan-glukanový komplex. Mechanické vlastnosti skafoldů byly upraveny síťováním pomocí karbodiimidů. Růstové faktory byly dodány formou destičkového lyzátu. Byl zkoumán vliv biologicky aktivních aditiv, siťovacího činidla a obohacení růstovými faktory na vlastnosti připravených skaffoldů a jejich bioaktivitu v tkáních živých organismů. Konkrétně byly studovány morfologické vlastnosti, struktura, porozita, botnání, stabilita, chemické složení, teplota denaturace a biologické vlastnosti. K charakterizaci byly použity metody rastrovací elektronová mikroskopie, infračervená spektroskopie, diferenční kompenzační kalorimetrie a konfokální mikroskop. Připravené kolagenové substráty obohaceny bioaktivním aditivem a destičkovým lyzátem mohou být využity v biomedicíně jako skafoldy pro růst buněk v systémech s nízkou mechanickou zátěží.The thesis deals with the preparation of 3D porous collagen scaffolds by freeze-drying and their modification with bioactive compounds. The natural polysaccharides, chitosan, calcium oxidized cellulose and chitin/chitosan-glucan complex for the modification have been used. The mechanical properties of the scaffolds have been enhanced by crosslinking process with carbodiimides. Growth factors have been delivered in the form of platelet lysate. The influence of biologically active additives, crosslinking agents, and enrichment with growth factors on the properties of the prepared scaffold and their bioactivity in tissues of living organisms have been investigated. Specifically, this study includes the morphological properties, structure, porosity, swelling stability, chemical composition, temperature of denaturation and biological properties. Scanning electron microscopy, infrared specktroscopy, differential scanning calorimetry and confocal microscopy have been used to the characterization. Prepared collagen substrates involving bioactive additive and platelet lysate could be used as scaffold for growing cells in systems with low mechanical loading and which has potential application in biomedicine.

Evaluation of a co-culture of rapidly isolated chondrocytes and stem cells seeded on tri-layered collagen-based scaffolds in a caprine osteochondral defect model

Cartilage has poor regenerative capacity and thus damage to the joint surfaces presents a major clinical challenge. Recent research has focussed on the development of tissue-engineered and cell-based approaches for the treatment of cartilage and osteochondral injuries, with current clinically available cell-based approaches including autologous chondrocyte implantation and matrix-assisted autologous chondrocyte implantation. However, these approaches have significant disadvantages due to the requirement for a two-stage surgical procedure and an in vitro chondrocyte expansion phase which increases logistical challenges, hospital times and costs. In this study, we hypothesized that seeding biomimetic tri-layered scaffolds, with proven regenerative potential, with chondrocyte/infrapatellar fat pad stromal cell co-cultures would improve their regenerative capacity compared to scaffolds implanted cell-free. Rapid cell isolation techniques, without the requirement for long term in vitro culture, were utilised to achieve co-cultures of chondrocytes and stromal cells and thus overcome the limitations of existing cell-based techniques. Cell-free and cell-seeded scaffolds were implanted in osteochondral defects, created within the femoral condyle and trochlear ridge, in a translational large animal goat model. While analysis showed trends towards delayed subchondral bone healing in the cell-seeded scaffold group, by the 12 month timepoint the cell-free and cell-seeded groups yield cartilage and bone tissue with comparable quality and quantity. The results of the study reinforce the potential of the biomimetic tri-layered scaffold to repair joint defects but failed to demonstrate a clear benefit from the addition of the CC/FPMSC co-culture to this scaffold. Taking into consideration the additional cost and complexity associated with the cell-seeded scaffold approach, this study demonstrates that the treatment of osteochondral defects using cell-free tri-layered scaffolds may represent a more prudent clinical approach

Nano-carbonate hydroxyapatite synthesis through CO2 absorption and wet precipitation

학위논문 (석사)-- 서울대학교 대학원 : 재료공학부, 2016. 2. 남기태.Calcium phosphate compounds are used as a bone implant material for the treatment of bond related disease or injury. Among many calcium phosphate compounds, hydroxyapatite (HAP, Ca10(PO4)6(OH)2) and β-tricalcium phosphate (β-TCP, Ca3(PO4)2) are the most widely used. Although the major components of natural bone are calcium and phosphate, there are many other ions in the bone such as carbonate, sodium, and kalium. Therefore, many studies about ion substitution in HAP were reported. Especially, the carbonate substituted hydroxyapatite is of great interest because of its composition similarity with natural bone and potential to enhance bioactivity of bone grafts. There are many carbonate hydroxyapatite synthesis methods, but most of them use carbon containing chemical for the source of substituted carbonate during the synthesis. In this research, nano-carbonate hydroxyapatite (CHA) was successfully synthesized through wet precipitation method in aqueous solution. By creating superoxide anion radical in the system, atmospheric CO2 was captured and used as a source of carbonate groups in CHA. CHA formation was confirmed using XRD and FT-IR analysis, and comparison study between synthesized CHA and HAP was carried out. The gas chromatography result showed that our CHA synthesis system absorbed large amount of CO2 in the air efficiently. After that, we observed phase transformation occurred during the CHA synthesis. The result showed that dicalcium phosphate dehydrate (DCPD) was intermediate phase of CHA. Lastly, we succeeded in controlling substituted CO3 amount in CHA by controlling used H2O2 amount. As a result, we successfully synthesized CHA with bone similar composition. This research has two major impacts. First of all, this synthesis method proposes a new way to mineralize large amount of atmospheric CO2 and use the mineralized form of CO2 as the carbon source of CHA. Second, synthesized CHA can be used as bone implant material because of its chemical composition similarity to natural bone. Consequently, our CHA synthesis system presents the innovative method for synthesizing bone implant material while capturing atmospheric CO2.Chapter 1. INTRODUCTION 1 1.1 Introduction of biominerals and calcium phosphate compounds as bond implant material 1 1.1.1 Chemical composition of human bone 1 1.1.2 Requirements for bone implant material 2 1.1.3 Synthetic calcium phosphate compounds as bone implant material: hydroxyapatite and β-tricalcium phosphate 3 1.2 Carbonate hydroxyapatite 7 1.2.1 Types of carbonate hydroxyapatite 7 1.2.2 Carbonate hydroxyapatite as bone implant material 11 1.2.3 Synthesis methods of carbonate hydroxyapatite 15 1.3 Carbon capture and storage (CCS) 16 1.3.1 Atmospheric CO2 concentration and current status 16 1.3.2 Carbon capture & storage methods 19 1.3.3 Air capture 22 1.4 Carbonation of metal hydroxide sorbents 24 1.4.1 Alkali/Alkali earth metal hydroxide sorbents 24 1.4.2 Superoxide effect 28 1.5 Research scope and design 30 Chapter 2. MATERIALS AND EXPERIMENTS 32 2.1 Sample preparation of carbonate hydroxyapatite 32 2.1.1 Materials 32 2.1.2 Synthesis of carbonate hydroxyapatite 32 2.1.3 Amount modification of substituted carbonate in carbonate hydroxyapatite 35 2.2 Characterization 37 2.2.1 Powder X-ray diffraction (XRD) 37 2.2.2 Field emission scanning electron microscopy (FESEM) 37 2.2.3 Fourier transform infrared spectroscopy (FT-IR) 38 2.2.4 Element analyzer 38 2.3 Carbonation confirmation 39 2.3.1 Electron paramagnetic resonance (EPR) 39 2.3.2 Gas chromatography (GC) 41 2.4 Stability of synthesized carbonate hydroxyapatite 41 2.4.1 Solubility test 41 Chapter 3. RESULT AND DISCUSSION 43 3.1 Material characterization 43 3.1.1 Characterization of carbonate hydroxyapatite 43 3.1.1.1 XRD and SEM studies 43 3.1.1.2 FT-IR studies 49 3.1.2 Solubility of carbonate hydroxyapatite 52 3.2 CO2 absorption during the process 55 3.2.1 CO2 absorption from the air 55 3.2.2 Confirmation of radical formation 58 3.3 Phase transformation during carbonate hydroxyapatite formation 61 3.3.1 Phase transformation during the synthesis 61 3.4 CO3 substituted amount control 70 3.4.1 H2O2 amount effect 70 Chapter 4. CONCLUSION 75 References 77 초 록 86Maste