Scaffold Mechanical Strength Research Articles

Oxygen-Generating Scaffolds for Cardiac Tissue Engineering Applications.

Homogeneous vascularization of implanted tissue constructs can extend to 5 weeks, during which cell death can occur due to inadequate availability of oxygen. Researchers are engineering biomaterials that generate and release oxygen in a regulated manner, in an effort to overcome this hurdle. A main limitation of the existing oxygen-generating biomaterials is the uncontrolled release of oxygen, which is ultimately detrimental to the cells. This study demonstrates the incorporation of calcium peroxide (CaO2) within a hydrophobic polymer, polycaprolactone (PCL), to yield composite scaffolds with controlled oxygen release kinetics sustained over 5 weeks. Oxygen-generating microparticles coencapsulated with cardiomyocytes in a gelatin-based hydrogel matrix can serve as model systems for cardiac tissue engineering. Specifically, the results reveal that the oxygen-generating microspheres significantly improve the scaffold mechanical strength ranging from 5 kPa to 35 kPa, have an average scaffold pore size of 50-100 μm, swelling ratios of 33.3-29.8%, and degradation with 10-49% remaining mass at the end of a 48 h accelerated enzymatic degradation. The oxygen-generating scaffolds demonstrate improvement in cell viability, proliferation, and metabolic activity compared to the negative control group when cultured under hypoxia. Additionally, the optimized oxygen-generating constructs display no cytotoxicity or apoptosis. These oxygen-generating scaffolds can possibly assist the in vivo translation of cardiac tissue constructs.

Fabrication, characterization, and optimization of a novel copper-incorporated chitosan/gelatin-based scaffold for bone tissue engineering applications.

Introduction: Fabricating composite scaffolds with improved physicochemical properties as artificial microenvironments are of great interest in bone tissue engineering. Given advantageous properties of nano-hydroxyapatite/chitosan/gelatin (nHA/Cs/Gel) scaffolds, the present study aimed to synthesize a modified nHA/Cs/Gel biomimetic scaffold with improved features. Methods: Pure and copper (Cu)-substituted nHA was synthesized using the chemical precipitation method under controlled pH and temperature. Pure and Cu-substituted nHA/Cs/Gel scaffolds were fabricated by salt-leaching/freeze-drying method. Physicochemical characteristics of nanoparticles and scaffolds were explored using XRD, FTIR, FE-SEM/EDX, and ICP. Besides, scaffold mechanical strength, degradation, porosity, swelling, biomineralization, and cytocompatibility were assessed. Results: Pure and Cu-substituted nHA were synthesized and characterized with appropriate Cu substitution and improved physical properties. All scaffolds were highly porous (porosity > 98%) and Cu incorporation reduced porosity from 99.555 ± 0.394% to 98.69 ± 0.80% while enlarged the pore size to more than100 µm. Cu-substitution improved the scaffold mechanical strength and the best result was observed in nHA.Cu5%/Cs/Gel scaffolds by the compressive strength 88.869 ± 19.574 MPa. Furthermore, 3% and 5% Cu-substituted nHA enhanced the scaffold structural stability and supported osteoblast spread, adhesion, survival, mineralization, and proliferation. Moreover, long-term and sustainable Cu release from scaffolds was observed within 28 days. Conclusion: Cu-substituted nHA/Cs/Gel scaffolds mimic the porous structure and mechanical strength of cancellous bone, along with prolonged degradation and Cu release, osteoblast attachment, viability, calcium deposition, and proliferation. Taken together, our results indicate the upgraded properties of nHA.Cu5%/Cs/Gel scaffolds for future applications in bone tissue engineering.

Evaluation of Detergent-Free and Detergent-Based Methods for Decellularization of Murine Skin.

Acute and chronic wounds contribute to increased morbidity and mortality in affected people and impose significant financial burdens on healthcare systems. For these challenging wounds, acellular dermal matrices (ADMs) have been used as a biological wound coverage. Unlike engineered dermal matrices, ADMs are prepared through the removal of cells from skin, while preserving the extracellular matrix structure and function. In this study, our primary objective was to develop a detergent-free method for decellularization of the skin to mitigate chemical stress on matrix molecules. Then, we performed a set of in vitro and in vivo experiments to compare this method with nonionic and anionic detergent methods. All decellularization methods satisfactorily removed cells and supported fibroblast growth and migration in vitro. Sulfated glycosaminoglycan content was reduced significantly (p < 0.05) only in the ionic detergent treatment group. In contrast to the detergent-free method, all detergent-based methods significantly reduced scaffold mechanical strength and elastin content (p < 0.05). Three weeks after transplantation, the results showed reepithelialization, angiogenesis, and migration of host cell into scaffolds with no induction of immunogenic reaction in all ADM groups tested. In our study, the detergent-free method showed better preservation of matrix composition and biomechanical properties, but after transplantation, all methods of ADM preparation resulted in equally biofunctional matrices as wound coverage.

Biomineralization of Recombinant Peptide Scaffolds: Interplay among Chemistry, Architecture, and Mechanics.

Biomineralized scaffolds are an attractive option for bone tissue engineering, being similar to native bone. However, optimization is difficult, due to the complex interplay among architecture, chemistry, and mechanics. Utilizing biomimetic nucleation, linear mineralized scaffolds were created from a collagen type I based recombinant peptide (RCP). Osteoblast mineralization was assessed, in response to changes in scaffold architecture, hydroxyapatite (HA) content, and mechanics. Changes in scaffold pore size (150-450 μm) had little effect on mRNA levels but influenced cell proliferation, achieving a balance between nutrient diffusion and surface area for cell attachment at 300 μm. Increasing the scaffold mechanical strength, from 2.9 to 5.2 kPa, enhanced the expression of osteocalcin, a late marker of mineralization. Further addition of HA, up to 20 wt %, increased osteoblast mineralization, without altering the compressive modulus. Thus, it was shown that architectural cues influence cellular proliferation, while the scaffold chemistry and mechanics independently contribute to gene expression.

Multilayered polycaprolactone/gelatin fiber-hydrogel composite for tendon tissue engineering

Regeneration of injured tendon and ligament (T&L) remains a clinical challenge due to their poor intrinsic healing capacity. Tissue engineering provides a promising alternative treatment approach to facilitate T&L healing and regeneration. Successful tendon tissue engineering requires the use of three-dimensional (3D) biomimetic scaffolds that possess the physical and biochemical features of native tendon tissue. We report here the development and characterization of a novel composite scaffold fabricated by co-electrospinning of poly-ε-caprolactone (PCL) and methacrylated gelatin (mGLT). We found that photocrosslinking retained mGLT, resulted in a uniform distribution of mGLT throughout the depth of scaffold and also preserved scaffold mechanical strength. Moreover, photocrosslinking was able to integrate stacked scaffold sheets to form multilayered constructs that mimic the structure of native tendon tissues. Importantly, cells impregnated into the constructs remained responsive to topographical cues and exogenous tenogenic factors, such as TGF-β3. The excellent biocompatibility and highly integrated structure of the scaffold developed in this study will allow the creation of a more advanced tendon graft that possesses the architecture and cell phenotype of native tendon tissues. Statement of SignificanceThe clinical challenges in tendon repair have spurred the development of tendon tissue engineering approaches to create functional tissue replacements. In this study, we have developed a novel composite scaffold as a tendon graft consisting of aligned poly-ε-caprolactone (PCL) microfibers and methacrylated gelatin (mGLT). Cell seeding and photocrosslinking between scaffold layers can be performed simultaneously to create cell impregnated multilayered constructs. This cell-scaffold construct combines the advantages of PCL nanofibrous scaffolds and photocrosslinked gelatin hydrogels to mimic the structure, mechanical anisotropy, and cell phenotype of native tendon tissue. The scaffold engineered here as a building block for multilayer constructs should have applications beyond tendon tissue engineering in the fabrication of tissue grafts that consist of both fibrous and hydrogel components.

Norfloxacin-loaded collagen/chitosan scaffolds for skin reconstruction: Preparation, evaluation and in-vivo wound healing assessment

Biomaterial scaffolds are versatile tools as drug carrier for treatment of wounds. A series of norfloxacin-loaded scaffolds were synthesized for treatment of wounds by combining collagen with two different types of chitosan using freeze-drying technique. Subsequently, scaffolds were screened in terms of morphology, water absorption and retention capacity, biodegradation, ex-vivo bioadhesive strength, in-vitro drug release biological compatibility, X-ray diffractometry, differential scanning calorimetry as well as in-vivo evaluation. The results indicate that the scaffold mechanical strength is dependent on the type of used chitosan. The prepared scaffolds contained interconnected porous architecture. The scaffolds had high water uptake and retention capacity with extended biodegradation rate. Scaffolds prepared with chitosan HCl showed superior bioadhesive strength compared to those prepared with low molecular weight chitosan. All scaffolds showed almost 100% drug release within 24h. As identified by the terahertz pulsed imaging measurements, there is single scaffold area with the same concentration. After 28days of wound dressing with selected norfoloxacin-loaded or unloaded collagen/chitosan scaffolds in Albino rats, it was found that the tissue regeneration time was fast compared to non-treated wounds. Furthermore, the drug-loaded scaffolds showed normal structure of an intact epidermal layer as well as the underlying dermis as revealed by histopathological studies. The obtained results suggest that the investigated norfloxacin-loaded collagen/chitosan scaffold is a potential candidate for skin regeneration application.

Functionalized carbon nanotube reinforced scaffolds for bone regenerative engineering: fabrication, in vitro and in vivo evaluation

Designing biodegradable scaffolds with bone-compatible mechanical properties has been a significant challenge in the field of bone tissue engineering and regenerative engineering. The objective of this work is to improve the polymeric scaffold's mechanical strength by compositing it with mechanically superior carbon nanotubes. Poly(lactide-co-glycolide) (PLGA) microsphere scaffolds exhibit mechanical properties in the range of human cancellous bone. On the other hand, carbon nanotubes have outstanding mechanical properties. The aim of this study is to improve further the mechanical strength of PLGA scaffolds such that they may be applicable for a wide range of load-bearing repair and regeneration applications. We have formed composite microspheres of PLGA containing pristine and modified (with hydroxyl (OH), carboxylic acid (COOH)) multi-walled carbon nanotubes (MWCNTs), and fabricated them into three-dimensional porous scaffolds. Results show that by adding only 3% MWCNTs, the compressive strength and modulus was significantly increased (35 MPa, 510.99 MPa) compared to pure PLGA scaffolds (19 MPa and 166.38 MPa). Scanning electron microscopy images showed excellent cell adhesion and proliferation. In vitro studies exhibited good cell viability, proliferation and mineralization. The in vivo study, however, indicated differences in inflammatory response throughout the 12 weeks of implantation, with OH-modified MWCNTs having the least response, followed by unmodified and COOH-modified exhibiting a more pronounced response. Overall, our results show that PLGA scaffolds containing water-dispersible MWCNTs are mechanically stronger and display good cellular and tissue compatibility, and hence are potential candidates for load-bearing bone tissue engineering.

Biocompatibility and strengthening of porous hydroxyapatite scaffolds using poly(l-lactic acid) coating

Hydroxyapatite (HA) is a well-known biocompatible bone substitute. Porous HA is more resorbable and osteoconductive compared with non-porous HA, and has been studied both experimentally and clinically. However, the mechanical strength of porous HA scaffolds is known to be weak. In this study, we developed a porous HA scaffold coated with a synthetic biodegradable polymer, poly(l-lactic acid) (PLLA), to strengthen the scaffold. PLLA-coated HA pellets were used to investigate the in vitro proliferation and alkaline phosphatase (ALP) activity of osteoblasts. PLLA-coated porous HA scaffolds were observed using scanning electron microscopy to investigate surface characteristics, porosity, and mechanical strength. PLLA coating concentration varied from 2 to 10 wt%. Osteoblast proliferation was higher in HA samples coated with PLLA compared with non-coated. ALP activity was highest on 8 wt% PLLA-coating after 3 days and on 4 wt% and 6 wt% PLLA after 9 and 12 days. Porous HA scaffolds with higher concentrations of PLLA were found to have a smoother, flatter surface. This enhanced proliferation and attachment of osteoblasts onto the porous HA scaffold. PLLA solution at a concentration of 10 wt% decreased scaffold porosity to half that of HA scaffolds with no PLLA coating. Scaffold mechanical strength was increased two-fold with a PLLA concentration of 2 wt%. Based on in vitro experimentation, it can be concluded that PLLA-coating on porous HA scaffolds enhances both the biocompatibility and the mechanical strength.

A novel strontium‐doped calcium polyphosphate/erythromycin/poly(vinyl alcohol) composite for bone tissue engineering

It is our goal to develop bactericidal bone scaffolds with osteointegration potential. In this study, poly(vinyl alcohol) (PVA) coating (7%) was applied to an erythromycin (EM)-impregnated strontium-doped calcium polyphosphate (SCPP) scaffold using a simple slurry dipping method. MicroCT analysis showed that PVA coating reduced the average pore size and the percentage of pore interconnectivity to some extent. Compressive strength tests confirmed that the PVA coating significantly increased material elasticity and slightly enhanced the scaffold mechanical strength. It was also confirmed that the PVA coatings allowed a sustained EM release that is controlled by diffusion through the intact PVA hydrogel layer, irrespective of the drug solubility. PVA coating did not inhibit the EM bioactivity when the scaffolds were immersed in simulated body fluid for up to 4 weeks. EM released from SCPP-EM-PVA composite scaffolds maintained its capability of bacterial growth (S. aureus) inhibition. PVA coating is biocompatible and nontoxic to MC3T3 preosteoblast cells. Furthermore, we found that SCPP-EM-PVA composite scaffolds and their eluants remarkably inhibited RANKL-induced osteoclastogenesis in a murine RAW 264.7 macrophage cell line. Thus, this unique multifunctional bioactive composite scaffold has the potential to provide controlled delivery of relevant drugs for bone tissue engineering.

Multi-functional P(3HB) microsphere/45S5 Bioglass®-based composite scaffolds for bone tissue engineering

Novel multi-functional P(3HB) microsphere/45S5 Bioglass®-based composite scaffolds exhibiting potential for drug delivery were developed for bone tissue engineering. 45S5 Bioglass®-based glass–ceramic scaffolds of high interconnected porosity produced using the foam-replication technique were coated with biodegradable microspheres (size < 2 μm) made from poly(3-hydroxybutyrate), P(3HB), produced using Bacillus cereus SPV. A solid-in-oil-in-water emulsion solvent extraction/evaporation technique was used to produce these P(3HB) microspheres. A simple slurry-dipping method, using a 1 wt.% suspension of P(3HB) microspheres in water, dispersed by an ultrasonic bath, was used to coat the scaffold, producing a uniform microsphere coating throughout the three-dimensional scaffold structure. Compressive strength tests confirmed that the microsphere coating slightly enhanced the scaffold mechanical strength. It was also confirmed that the microsphere coating did not inhibit the bioactivity of the scaffold when immersed in simulated body fluid (SBF) for up to 4 weeks. The hydroxyapatite (HA) growth rate on P(3HB) microsphere-coated 45S5 Bioglass® composite scaffolds was very similar to that on the uncoated control sample, qualitatively indicating similar bioactivity. However, the surface topography of the HA surface layer was affected as shown by results obtained from white light interferometry. The roughness of the surface was much higher for the P(3HB) microsphere-coated scaffolds than for the uncoated samples, after 7 days in SBF. This feature would facilitate cell attachment and proliferation. Finally, gentamycin was successfully encapsulated into the P(3HB) microspheres to demonstrate the drug delivery capability of the scaffolds. Gentamycin release kinetics was determined using liquid chromatography–mass spectrometry. The release of the drug from the coated composite scaffolds was slow and controlled when compared to the observed fast and relatively uncontrolled drug release from the bone scaffold (without microsphere coating). Thus, this unique multifunctional bioactive composite scaffold has the potential to enhance cell attachment and to provide controlled delivery of relevant drugs for bone tissue engineering.

Electrospinning of synthesized triblock copolymers of ε-caprolactone and L-lactide for the application of vascular tissue engineering

Biodegradable triblock copolymers of ε-caprolactone and L-lactide with varying compositions and molecular weights have been synthesized. They were then used to fabricate compliant small-diameter tissue engineered vascular scaffolds by using an electrospinning technique. The in vitro and in vivo degradation of the ultrafine fabrics was monitored to be faster than their counterpart cast films. A favorable interaction between the scaffolds and the mouse fibroblast L929 cells was demonstrated via MTT assay. A confluent, adherent monolayer of canine mesenchymal stem cells was observed in the tubular scaffold lumen after culture in a bioreactor for 3 days. The scaffold mechanical strength was strong enough to be transplanted into the canine carotid artery.

Advanced Mechanical Properties of Porous Hydroxyapatite Ceramics Prepared Using Hydroxyapatite Slip and its Slurry Synthesis

There is a clinical need for synthetic scaffolds that will promote bone regeneration. Important factors include obtaining an optimal porosity and size of interconnecting macropores whilst maintaining scaffold mechanical strength, enabling complete penetration of cells and nutrients throughout the scaffold, preventing the formation of necrotic tissue in the centre of the scaffold. To address this we investigated flexural strength of bimodal porous apatite ceramics prepared using apatite slurry and its slurry synthesis was studied. Slips with different contents of HAp (K-HAp and T-HAp) and deflocculant were prepared by milling in a pot mill. The viscosity of slurries made of commercial T-HAp powder showed a drop after 3 hours’ milling, but the viscosity of slurry with high solid content of k-HAp and 2.0 wt% deflocculant increased with an increase of milling time after 2 hours’ milling. The porosity and flexural strength of the porous HAp prepared by heating the foam dipped in K-HAp slip with 2.0 wt% of deflocculant and 0.5wt% of foaming regent heated at 1200°C were 62.4 % and 14.7 MPa, and those in T-HAp were 59.7 % and 15.2 MPa with 1.5 wt% of deflocculant and 0.5wt% of foaming regent heated at 1200°C.

The influence of dispersant concentration on the pore morphology of hydroxyapatite ceramics for bone tissue engineering

There is a clinical need for synthetic scaffolds that will promote bone regeneration. Important factors include obtaining an optimal porosity and size of interconnecting windows whilst maintaining scaffold mechanical strength, enabling complete penetration of cells and nutrients throughout the scaffold, preventing the formation of necrotic tissue in the centre of the scaffold. To address this we investigated varying slip deflocculation in order to control the resulting porosity, pore size and interconnecting window size whilst maintaining mechanical strength. Hydroxyapatite (HA) porous ceramics were prepared using a modified slip casting process. Rheological measurements of the HA slips were used to identify deflocculation conditions which resulted in changes in the cell and window sizes of the resulting ceramics. Sintered ceramics were characterised by X-ray diffraction (XRD) and scanning electron microscopy (SEM). Pore and window size distribution was determined by SEM. XRD analysis confirmed that the crystal structure remained HA after the sintering process. SEM showed that HA porous ceramics presented a highly interconnected porous network with average pore sizes ranging from 391±39 to 495±25 μm. The average window size varied from 73±5 to 135±7 μm. Pore diameters obtained were controllable in the range 200–500 μm. Window sizes were in the range 30–250 μm. The use of dispersant concentration allows pore and window size to be modified whilst maintaining control over porosity demonstrated by a porosity of 85% for seven different dispersant concentrations. The advantage of this approach allows the correlation between the rheological conditions of the slip and the resultant sintered ceramic properties. In particular, optimising the ceramic strength by controlling the agglomeration during the casting process.