Scaffold Volume Research Articles

Efficient fabrication of 3D bioprinted functional sensory neurons using an inducible Neurogenin-2 human pluripotent stem cell line

Three-dimensional (3D) tissue models have gained recognition for their improved ability to mimic the native cell microenvironment compared to traditional two-dimensional models. This progress has been driven by advances in tissue-engineering technologies such as 3D bioprinting, a promising method for fabricating biomimetic living tissues. While bioprinting has succeeded in generating various tissues to date, creating neural tissue models remains challenging. In this context, we present an accelerated approach to fabricate 3D sensory neuron (SN) structures using a transgenic human pluripotent stem cell (hPSC)-line that contains an inducible Neurogenin-2 (NGN2) expression cassette. The NGN2 hPSC line was first differentiated to neural crest cell (NCC) progenitors, then incorporated into a cytocompatible gelatin methacryloyl-based bioink for 3D bioprinting. Upregulated NGN2 expression in the bioprinted NCCs resulted in induced SN (iSN) populations that exhibited specific cell markers, with 3D analysis revealing widespread neurite outgrowth through the scaffold volume. Calcium imaging demonstrated functional activity of iSNs, including membrane excitability properties and voltage-gated sodium channel (NaV) activity. This efficient approach to generate 3D bioprinted iSN structures streamlines the development of neural tissue models, useful for the study of neurodevelopment and disease states and offering translational potential.

3D-printed Ti6Al4V thoracic fusion cage: Biomechanical behavior and strengthening mechanism

To effectively replace diseased and damaged thoracic vertebrae, a series of three-dimensional (3D) printed titanium (Ti) alloy (Ti6Al4V) thoracic fusion cages with varying volume ratio of reinforced scaffolds are designed, and their structure-function relationships are investigated systematically. It is demonstrated that the mechanical strength of fusion cage is gradually enhanced as the scaffold volume increases, which may be attributed to its textural transformation from α phase to β phase. The compressive, shear, and fatigue of fusion cages successfully attain the International Organization for Standardization (ISO 23089), when the scaffold volume ratio exceeds 15%. Furthermore, the biomechanical analyses, including stress distributions and subsidence in flexion, extension, axial rotation, and lateral bending sections of fusion cages, further shed light on their potential clinical applications. This work deeply deciphers the intrinsic connection between biomechanical property and material texture of the thoracic fusion cages, offering valuable insights into their strengthening mechanism, which providing a guidance for the precise design and quick synthesis of other 3D-printed Ti6Al4V implants.

Perfusable Biohybrid Designs for Bioprinted Skeletal Muscle Tissue.

Engineered, centimeter-scale skeletal muscle tissue (SMT) can mimic muscle pathophysiology to study development, disease, regeneration, drug response, and motion. Macroscale SMT requires perfusable channels to guarantee cell survival, and support elements to enable mechanical cell stimulation and uniaxial myofiber formation. Here, stable biohybrid designs of centimeter-scale SMT are realized via extrusion-based bioprinting of an optimized polymeric blend based on gelatin methacryloyl and sodium alginate, which can be accurately coprinted with other inks. A perfusable microchannel network is designed to functionally integrate with perfusable anchors for insertion into a maturation culture template. The results demonstrate that i) coprinted synthetic structures display highly coherent interfaces with the living tissue, ii) perfusable designs preserve cells from hypoxia all over the scaffold volume, iii) constructs can undergo passive mechanical tension during matrix remodeling, and iv) the constructs can be used to study the distribution of drugs. Extrusion-based multimaterial bioprinting with the inks and design realizes in vitro matured biohybrid SMT for biomedical applications.

Is three-dimension-printed mesh scaffold an alternative to reconstruct cavity bone defects near joints?

Reconstruction of cavity bone defects after curettage of benign bone tumours around the joint remains challenging. We designed a novel 3D-printed mesh scaffold as a substitute for bone cement, aiming to support the articular surface, protect the subchondral bone, and reduce complication rates. We retrospectively analyzed seven patients who received curettage and reconstruction using a 3D-printed mesh scaffold between January 2020 and June 2021. Pain and function were evaluated using the 10-cm Visual Analogue Scale (VAS) score and the 1993 version of the Musculoskeletal Tumor Society (MSTS-93) score. Radiographs were used to evaluate articular surface supporting, subchondral bone protection, and complications. The median functional MSTS-93 and VAS scores were both improved after surgery, and the median 3D-printed mesh scaffold volume was smaller than the median defect volume. Articular surface supporting, subchondral bone preservation, and osteogenesis were observed post-operatively. No related complications were observed during the last follow-up. The 3D-printed mesh scaffold provided sufficient mechanical support for the articular surface and protected the subchondral bone. We recommended the 3D-printed mesh structure as an alternative to repair cavity bone defects around joints.

Mini- and macro-scale direct perfusion bioreactors with optimized flow for engineering 3D tissues.

Bioreactors enabling direct perfusion of cell suspensions or culture media through the pores of 3D scaffolds have long been used in tissue engineering to improve cell seeding efficiency as well as uniformity of cell distribution and tissue development. A macro-scale U-shaped bioreactor for cell culture under perfusion (U-CUP) has been previously developed. In that system, the geometry of the perfusion chamber results in rather uniform flow through most of the scaffold volume, but not in the peripheral regions. Here, the design of the perfusion chamber has been optimized to provide a more homogenous perfusion flow through the scaffold. Then, the design of this macro-scale flow-optimized perfusion bioreactor (macro-Flopper) has been miniaturized to create a mini-scale device (mini-Flopper) compatible with medium-throughput assays. Computational fluid dynamic (CFD) modeling of the new chamber design, including a porous scaffold structure, revealed that Flopper bioreactors provide highly homogenous flow speed, pressure, and shear stress. Finally, a proof-of-principle of the functionality of the Flopper systems by engineering endothelialized stromal tissues using human adipose tissue-derived stromal vascular fraction (SVF) cells has been offered. Preliminary evidence showing that flow optimization improves cell maintenance in the engineered tissues will have to be confirmed in future studies. In summary, two bioreactor models with optimized perfusion flow and complementary sizes have been proposed that might be exploited to engineer homogenous tissues and, in the case of the mini-Flopper, for drug testing assays with a limited amount of biological material.

Stimulation of MSCs Osteogenic Differentiation on a Micro/ Nanofibrous Scaffold Using Mechanical Stimulation in the Perfusion Bioreactor

In tissue engineering, the 3D constructs seeded with cells tested in vitro in static conditions suffer from poor cellular penetration and non-homogenous matrix deposition, due to the insufficient flow of nutrients, gases and signal molecules through the scaffold volume. However, a dynamic culture of cells can improve these drawbacks and simulate physiological conditions, such as flow in the canalicular system of bones. The purpose of this comparative study was to evaluate the different flow rates of 1 and 3 mL/min of perfusion bioreactor on human mesenchymal stromal cells (hMSCs), seeded in an osteogenic media on 3D porous constructs. After three weeks of dynamic cultivation, the metabolic activity and proliferation of the cells was evaluated, as well as the alkaline phosphatase (ALP) activity and the expression of bone related genes, RunX2, collagen type I and osteocalcin. The dynamic culture conditions did not influence the metabolic activity of hMSCs. The highest cell proliferation was detected on the scaffold cultured at a flow rate of 3 mL/min. The dynamic culture did not prove the penetration of cells into the scaffold's volume. The ALP activity, and PCR analysis of osteogenic markers RunX2 and collagen type I were highest on the scaffolds cultured at 1 mL/min, compared to the static culture and even to the dynamic culture at 3 mL/min. The study shows that hMSCs behave differently in different flow rates. While the higher flow rate (3 mL/min, shear stress 479 mPa) improved cell proliferation, the slower flow rate of 1 mL/min (shear stress 160 mPa) supported the ALP activity and expression of osteogenic marker genes. Interestingly, our expectation that dynamic culture improves cell penetration into the scaffold was not proved.
 
 
 
 
 
 
 
 
 
 
 
 
 

Integration of micro-CT and histology data for vasculature morpho-functional analysis in tissue regeneration

The demand for artificial or bioartificial engineered tissues is increasing today in regenerative medicine techniques to replace and restore the physiological function of damaged tissues. Such engineered constructs hold different properties depending on the tissue to be replicated. As for vascularized tissues, complex biocompatible structures, namely scaffolds, play a key role in supporting oxygen and nutrient supply, thus sustaining tissue neoformation and integration with the host. Scaffold architecture significantly impacts its regenerative potential, while preclinical trials are essential to define scaffold-host interactions. In compliance with the 3 R principle, there is a clear need to optimize both the procedures to evaluate scaffold performance and the analysis methodology decreasing the number of animals required to gain consistent data. In parallel, current technologies used in preclinical research generate huge amounts of data that need to be elaborated and interpreted correctly. Therefore, we designed this study to evaluate the results of scaffold integration with the host tissue after implantation in a mouse subcutaneous pocket model. We evaluated the angiogenic response developed by the host and the degree of scaffold integration by using a combined morphometric approach based on both histological and micro-CT analyses. Six-layer scaffolds, made of polycaprolactone (PCL) microspheres, with an ordered structure were produced by thermal sintering. Scaffolds were then implanted in BALB/c mice and retrieved 21 days post-implantation when the animals were deeply anesthetized and perfused with Microfil, a contrast agent for micro-CT. Here, we describe a method to extract quantitative data from micro-CT reconstructions such as (i) total vessel volume; (ii)% of vessel penetration; (iii) distribution of vessel diameters. The general principle of this approach is the refinement of the region of interest (ROI), thus producing a volume of interest (VOI) that matches scaffold volume. This VOI serves as a dataset from which to extract volumetric information. Then VOIs are divided into three identical parts, proximal, median, and distal, to follow the vessel progression into the scaffold, thus obtaining their depth of penetration (DoP). By this methodology, we observed in mean, among the analyzed samples, a vessel invasion for 1,38 mm3 corresponding to the 1,53% of the scaffold volume. We then looked at the diameter distribution being this value a key indicator of vessel maturity, highlighting that 55% of vessels fall into the range from 5,99–53.99 µm while the remaining 45% are distributed into intervals from 54 to 136 µm. In parallel, to evaluate tissue integration in detail, histological and immunofluorescent analyses were performed to look at vessel distribution and collagen synthesis. Histological results strongly correlate with the micro-CT data providing, however, an overview of the ingrowth tissues. In addition, by immunofluorescent analysis we demonstrate that newly formed vessels are mature at the considered time point and tissue collagen deposition is widespread within the scaffolds. Collectively, we propose a new method to track vessel formation by using a multi-modal approach posing the basis for: i) the fabrication of novel scaffolds for Tissue Engineering; ii) the integration of detailed information for a wide range of morphological and functional analyses.

Comparison of degradation behavior and osseointegration of 3D powder-printed calcium magnesium phosphate cement scaffolds with alkaline or acid post-treatment.

Due to the positive effects of magnesium substitution on the mechanical properties and the degradation rate of the clinically well-established calcium phosphate cements (CPCs), calcium magnesium phosphate cements (CMPCs) are increasingly being researched as bone substitutes. A post-treatment alters the materials’ physical properties and chemical composition, reinforcing the structure and modifying the degradation rate. By alkaline post-treatment with diammonium hydrogen phosphate (DAHP, (NH4)2HPO4), the precipitation product struvite is formed, while post-treatment with an acidic phosphate solution [e.g., phosphoric acid (PA, H3PO4)] results in precipitation of newberyite and brushite. However, little research has yet been conducted on newberyite as a bone substitute and PA post-treatment of CMPCs has not been described in the accessible literature so far. Therefore, in the present study, the influence of an alkaline (DAHP) or acid (PA) post-treatment on the biocompatibility, degradation behavior, and osseointegration of cylindrical scaffolds (h = 5.1 mm, Ø = 4.2 mm) produced from the ceramic cement powder Ca0.75Mg2.25(PO4)2 by the advantageous manufacturing technique of three-dimensional (3D) powder printing was investigated in vivo. Scaffolds of the material groups Mg225d (DAHP post-treatment) and Mg225p (PA post-treatment) were implanted into the cancellous part of the lateral femoral condyles in rabbits. They were evaluated up to 24 weeks by regular clinical, X-ray, micro-computed tomographic (µCT), and histological examinations as well as scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) analysis and compared with tricalcium phosphate (TCP). All materials showed excellent biocompatibility and rapid osseointegration. While TCP degraded only slightly, the CMPCs showed almost complete degradation. Mg225d demonstrated significantly faster loss of form and demarcability from surrounding bone, scaffold volume reduction, and significantly greater degradation on the side towards the bone marrow than to the cortex than Mg225p. Simultaneously, numerous bone trabeculae have grown into the implantation site. While these were mostly located on the side towards the cortex in Mg225d, they were more evenly distributed in Mg225p and showed almost the same structural characteristics as physiological bone after 24 weeks in Mg225p. Based on these results, the acid post-treated 3D powder-printed Mg225p is a promising degradable bone substitute that should be further investigated.

Travelling-Wave and Asymptotic Analysis of a Multiphase Moving Boundary Model for Engineered Tissue Growth

We derive a multiphase, moving boundary model to represent the development of tissue in vitro in a porous tissue engineering scaffold. We consider a cell, extra-cellular liquid and a rigid scaffold phase, and adopt Darcy’s law to relate the velocity of the cell and liquid phases to their respective pressures. Cell–cell and cell–scaffold interactions which can drive cellular motion are accounted for by utilising relevant constitutive assumptions for the pressure in the cell phase. We reduce the model to a nonlinear reaction–diffusion equation for the cell phase, coupled to a moving boundary condition for the tissue edge, the diffusivity being dependent on the cell and scaffold volume fractions, cell and liquid viscosities and parameters that relate to cellular motion. Numerical simulations reveal that the reduced model admits three regimes for the evolution of the tissue edge at large time: linear, logarithmic and stationary. Employing travelling-wave and asymptotic analysis, we characterise these regimes in terms of parameters related to cellular production and motion. The results of our investigation allow us to suggest optimal values for the governing parameters, so as to stimulate tissue growth in an engineering scaffold.

Integration of polyurethane meniscus scaffold during ACL revision is not reliable at 5years despite favourable clinical outcome.

The aim of this study was to evaluate the clinical outcome at 5-year follow-up of a one-step procedure combining anterior cruciate ligament (ACL) reconstruction and partial meniscus replacement using a polyurethane scaffold for the treatment of symptomatic patients with previously failed ACL reconstruction and partial medial meniscectomy. Moreover, the implanted scaffolds have been evaluated by MRI protocol in terms of morphology, volume, and signal intensity. Twenty patients with symptomatic knee laxity after failed ACL reconstruction and partial medial meniscectomy underwent ACL revision combined with polyurethane-based meniscal scaffold implant. Clinical assessment at 2- and 5-year follow-ups included VAS, Tegner Activity Score, International Knee Documentation Committee (IKDC), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), and the Lysholm Score. MRI evaluation of the scaffold was performed according to the Genovese scale with quantification of the scaffold's volume at 1- and 5-year follow-ups. All scores revealed clinical improvement as compared with the preoperative values at the 2- and 5-year follow-ups. However, a slight, but significant reduction of scores was observed between 2 and 5years. Concerning the MRI assessment, a significant reduction of the scaffold's volume was observed between 1 and 5years. Genovese Morphology classification at 5years included two complete resorptions (Type 3) and all the remaining patients had irregular morphology (Type 2). With regard to the Genovese Signal at the 5-year follow-up, three were classified as markedly hyperintense (Type 1), 15 as slightly hyperintense (Type 2), and two as isointense (Type 1). Simultaneous ACL reconstruction and partial meniscus replacement using a polyurethane scaffold provides favourable clinical outcomes in the treatment of symptomatic patients with previously failed ACL reconstruction and partial medial meniscectomy at 5years. However, MRI evaluation suggests that integration of the scaffold is not consistent. Level IV.

3D gel-printed porous magnesium scaffold coated with dibasic calcium phosphate dihydrate for bone repair in vivo.

Background/Objective: The treatment of bone defect has always been a difficult problem in orthopedic clinic. The search for alternative biodegradable implants is a hot topic. The development of biodegradable magnesium scaffolds for the treatment of bone defects has long been a goal of the public.MethodsIn this study, we proposed a porous magnesium scaffold prepared by 3D gel printing and surface modification with an additional calcium phosphate coating and use of its strength, degradability and slow degradation rate in a bone graft substitute material. The porous magnesium granular scaffold was prepared by 3D gel printing technology and modified by DCPD (Dibasic Calcium Phosphate Dihydrate) coating. The biocompatibility, degradation rate, and osteogenic ability of the scaffold were evaluated in vitro and in vivo.ResultsThe biocompatibility, in vivo degradation and bone defect healing response of the implants were investigated. Porous magnesium scaffolds were successfully prepared, and the strength of sintered scaffolds reached 5.38 ​MPa. The degradation rates of scaffolds were significantly reduced after coating with DCPD. The cell compatibility evaluation showed that DCPD-coated Mg scaffold was suitable for cell proliferation. In vivo biosafety monitoring showed that scaffold implantation did not cause an increase in Mg ion concentration in vivo, and no toxic damage was detected in the liver or kidney. Micro-CT and pathological results showed that a large amount of new bone was formed at 6 weeks. At 12 weeks, approximately 52% of the scaffold volume remained. At 24 weeks, osteogenesis, which was stimulated by some residual scaffold, still can be observed. In summary, this study suggests that 3D gel-printed DCPD-coated porous magnesium scaffolds have great potential as bone graft alternatives.ConclusionIn summary, this study suggests that 3D gel-printed DCPD-coated porous magnesium scaffolds have great potential as bone graft alternatives.The Translational potential of this articleThe translational potential of this article is to make use of the advantages of 3D gel printing technology with higher efficiency and lower cost compared with SLM and SLS technologies, and use pure magnesium powder as raw material to prepare degradable porous magnesium metal scaffolds, opening up a new technical route for the preparation of degradable porous magnesium scaffolds which are made for bone defect regeneration in the future.

Transforming the Degradation Rate of β-tricalcium Phosphate Bone Replacement Using 3-Dimensional Printing.

β-Tricalcium phosphate (β-TCP) is one of the most common synthetic bone grafting materials utilized in craniofacial reconstruction; however, it is limited by a slow degradation rate. The aim of this study was to leverage 3-dimensional (3D) printing in an effort to accelerate the degradation kinetics of β-TCP. Twenty-two 1-month-old New Zealand white rabbits underwent creation of calvarial and alveolar defects, repaired with 3D-printed β-TCP scaffolds coated with 1000 μM of osteogenic agent dipyridamole. Rabbits were euthanized after 2, 6, and 18 months after surgical intervention. Bone regeneration, scaffold degradation, and bone mechanical properties were quantified. Histological analysis confirmed the generation of vascularized and organized bone. Microcomputed tomography analysis from 2 to 18 months demonstrated decreased scaffold volume within calvarial (23.6% ± 2.5%, 5.1% ± 2.2%; P < 0.001) and alveolar (21.5% ± 2.2%, 0.2% ± 1.9%; P < 0.001) defects, with degradation rates of 54.6%/year and 90.5%/year, respectively. Scaffold-inducted bone generation within the defect was volumetrically similar to native bone in the calvarium (55.7% ± 6.9% vs 46.7% ± 6.8%; P = 0.064) and alveolus (31.4% ± 7.1% vs 33.8% ± 3.7%; P = 0.337). Mechanical properties between regenerated and native bone were similar. Our study demonstrates an improved degradation profile and replacement of absorbed β-TCP with vascularized, organized bone through 3D printing and addition of an osteogenic agent. This novel additive manufacturing and tissue engineering protocol has implications to the future of craniofacial skeletal reconstruction as a safe and efficacious bone tissue engineering method.

Transforming the Degradation Rate of β-tricalcium Phosphate Bone Replacement Using 3D Printers

Statement of the problemCraniofacial injuries often require resection of necrotic hard tissue. For large bone defects, autogenous bone grafting is ideal, but as is the case with all grafting procedures, is subject to limitations on quantity. Synthetic biomaterial driven three-dimensional (3D) printing technology of solid-free form fabrication (SFF) offers an alternative engineered healing approach. This research hypothesizes that a bioactive calcium-phosphate scaffold may successfully regenerate craniofacial bony defects in vivo, and that this newly formed bone will have mechanical properties similar to native bone as healing time elapses in vivo. The aim of this study was to leverage 3D printing of β-tricalcium phosphate (β-TCP), a common synthetic bone grafting material, to accelerate degradation kinetics of β-TCP while allowing for optimal bone regeneration/remodeling and bone mechanical properties. Materials and methodsTwenty-two 1-month-old New Zealand White rabbits underwent creation of calvarial and alveolar defects, repaired with 3D-printed β-TCP scaffolds coated with 1,000 μM of osteogenic agent dipyridamole. Rabbits were euthanized after 2, 6, and 18 months post-surgical intervention. Bone regeneration, scaffold degradation, and bone mechanical properties were quantified. ResultsHistology analysis confirmed the generation of vascularized and organized bone. Micro CT analysis from 2 to 18 months, demonstrated decreased scaffold volume within calvarial (23.6% ±2.5%, 5.1%±2.2%; P < .001) and alveolar (21.5%±2.2%, 0.2%±1.9%; P < .001) defects, with degradation rates 54.6%/year and 90.5%/year, respectively. Scaffold-inducted bone generation within the defect was volumetrically similar to native bone in the calvarium (55.7%±6.9% vs. 46.7%±6.8%; P = .064) and alveolus (31.4%±7.1% vs. 33.8%±3.7%; P = .337). Mechanical properties between regenerated and native bone were similar. Outcomes and conclusionThis study demonstrates an improved degradation profile and replacement of absorbed β-TCP with vascularized, organized bone through 3D printing, and the addition of an osteogenic agent. This novel additive manufacturing and tissue-engineering protocol has implications for the future of craniofacial skeletal reconstruction as a safe and efficacious bone tissue-engineering method.

4: Bone Tissue Engineering in the Growing Skull: A Short-term Study Using Dipyridamole-loaded 3D-printed Beta-tricalcium Phosphate Scaffolds to Repair Critically Sized Calvarial Defects in a Translational Animal Model

Purpose:The repair of bony defects remains a challenge to reconstructive surgeons due to limitations of current bone replacement techniques. Previous studies have shown that dipyridamole-loaded 3D-printed bioceramic (DIPY-3DPBC) scaffolds composed of 100% beta-tricalcium phosphate regenerate bone across critically sized defects in multiple in vivo models. However, a critical step to translating this bone tissue engineering construct to bedside is its successful application in a pre-clinical translational model that mimics biologic similarities to humans. Pigs are considered to be an excellent species for bone tissue engineering studies due to their similarities to humans both in bone biology and wound healing. In this short-term study, our group implanted DIPY-3DPBC scaffolds in an effort to repair critically sized bony defects in a translational, skeletally immature, growing craniofacial pig model. The purpose was to evaluate the performance of these 3D-printed constructs in terms of bone growth and scaffold absorption.Methods:Unilateral calvarial defects were created in six-week-old Göttingen minipigs (n=12). Four defects were left empty to serve as negative controls, four defects were filled with a 1000μM DIPY-3DPBC scaffold with a closed scaffold design (contains a solid barrier on the ectocortical side of the scaffold to prevent soft tissue infiltration), and four defects were filled with a 1000μM DIPY-3DPBC scaffold with an open design (no solid barrier on the ectocortical side). Animals were euthanized at 12-weeks post-operatively. Calvaria were subjected to micro-computed tomography (CT) for volumetric (3D) analysis. 3D reconstruction was performed using Amira software to quantitatively assess percent bone volume occupying the defect space (bone volume/total volume x 100%) and percent scaffold volume occupying the defect space (scaffold volume/total volume x 100%). A generalized linear mixed model (GLMM) was used to determine significance between groups. Results are presented using means with corresponding 95% confidence intervals and p-values.Results:Significantly higher mean (± 95% CI) percent bone volume was observed in calvarial defects treated with the scaffolds compared to negative controls (42.6% ± 8.6% vs 4.6% ± 12.1%, p≤0.001). Defects that were filled with closed cap scaffolds had a significantly greater mean percent bone volume compared to defects filled with the open scaffolds (52.9% ± 12.1% vs 32.3% ± 12.1%, p=0.024). No significant difference was detected between percent scaffold volume between closed and open scaffolds (4.4% ± 1.6% vs 3.7% ± 1.6%, p=0.493).Conclusions:The results from this work indicate an effective bone tissue engineering scaffold design that is able to regenerate bone across critically sized calvarial defects in a short-term skeletally immature, growing translational pig model.

Surface-Modified Poly(l-lactide-co-glycolide) Scaffolds for the Treatment of Osteochondral Critical Size Defects-In Vivo Studies on Rabbits.

Poly(l-lactide-co-glycolide) (PLGA) porous scaffolds were modified with collagen type I (PLGA/coll) or hydroxyapatite (PLGA/HAp) and implanted in rabbits osteochondral defects to check their biocompatibility and bone tissue regeneration potential. The scaffolds were fabricated using solvent casting/particulate leaching method. Their total porosity was 85% and the pore size was in the range of 250–320 µm. The physico-chemical properties of the scaffolds were evaluated using scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), X-ray diffractometry (XRD), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR), sessile drop, and compression tests. Three types of the scaffolds (unmodified PLGA, PLGA/coll, and PLGA/HAp) were implanted into the defects created in New Zealand rabbit femoral trochlears; empty defect acted as control. Samples were extracted after 1, 4, 12, and 26 weeks from the implantation, evaluated using micro-computed tomography (µCT), and stained by Masson–Goldner and hematoxylin-eosin. The results showed that the proposed method is suitable for fabrication of highly porous PLGA scaffolds. Effective deposition of both coll and HAp was confirmed on all surfaces of the pores through the entire scaffold volume. In the in vivo model, PLGA and PLGA/HAp scaffolds enhanced tissue ingrowth as shown by histological and morphometric analyses. Bone formation was the highest for PLGA/HAp scaffolds as evidenced by µCT. Neo-tissue formation in the defect site was well correlated with degradation kinetics of the scaffold material. Interestingly, around PLGA/coll extensive inflammation and inhibited tissue healing were detected, presumably due to immunological response of the host towards collagen of bovine origin. To summarize, PLGA scaffolds modified with HAp are the most promising materials for bone tissue regeneration.

Three-Dimensionally-Printed Bioactive Ceramic Scaffolds: Construct Effects on Bone Regeneration.

The utilization of three-dimensionally (3D)-printed bioceramic scaffolds composed of beta-tricalcium phosphate in conjunction with dipyridamole have shown to be effective in the osteogenesis of critical bone defects in both skeletally immature and mature animals. Furthermore, previous studies have proven the dura and pericranium's osteogenic capacity in the presence of 3D-printed scaffolds; however, the effect galea aponeurotica on osteogenesis in the presence of 3D scaffolds remains unclear. Critical-sized (11 mm) bilateral calvarial defects were created in 35-day old rabbits (n = 7). Two different 3D scaffolds were created, with one side of the calvaria being treated with a solid nonporous cap and the other with a fully porous cap. The solid cap feature was designed with the intention of preventing communication of the galea and the ossification site, while the porous cap permitted such communication. The rabbits were euthanized 8 weeks postoperatively. Calvaria were analyzed using microcomputed tomography, 3D reconstruction, and nondecalcified histologic sectioning in order assess differences in bone growth between the two types of scaffolding. Scaffolds with the solid (nonporous) cap yielded greater percent bone volume (P = 0.012) as well as a greater percent potential bone (P = 0.001) compared with the scaffolds with a porous cap. The scaffolds with porous caps also exhibited a greater percent volume of soft tissue (P < 0.001) presence. There were no statistically significant differences detected in scaffold volume. A physical barrier preventing the interaction of the galea aponeurotica with the scaffold leads to significantly increased calvarial bone regeneration in comparison with the scaffolds allowing for this interaction. The galea's interaction also leads to more soft tissue growth hindering the in growth of bone in the porous-cap scaffolds.

Assessment of the morphology and dimensional accuracy of 3D printed PLA and PLA/HAp scaffolds

IntroductionIn complex clinical conditions when physiological bone regeneration is insufficient, there is a need to develop synthetic material-based scaffolds. The morphologic properties of porous scaffolds are of crucial importance. The dimensional accuracy of 3D printed scaffolds can be affected by a variety of factors. Materials and methodsThree groups of 3D printed scaffolds were investigated: PLA1 (pure polylactic acid) printed with an FDM Ultimaker Original printer, PLA2 and composite PLA/hydroxyapatite (PLA/HAp) scaffolds printed with a Pharaoh XD 20. PLA/HAp filament was created with hot-melt extrusion (HME) equipment. The morphology of the prepared scaffolds was investigated with SEM, micro-CT and superimposition techniques, gravimetric and liquid displacement methods. ResultsLayer heights of PLA1 scaffolds varied the most. PLA1 scaffold volume statistically significantly differed from PLA2 (p < 0.001) and PLA/HAp (p < 0.01) groups. Filament composition had no effect on the volumes of the scaffolds printed with the Pharaoh XD 20 printer (p > 0.05). The total porosity of printed PLA/HAp scaffolds deviated the least from the original STL model. ConclusionsThis study showed that PLA/10% HAp filament fabricated with HME and printed with FFF 3D printer produced equal or even better accuracy of printed scaffolds than scaffolds printed with pure PLA filament. Further research is needed to analyze the effect of HAp on 3D scaffold morphology, accuracy, mechanical and biologic properties.

Gelatin coating increases in vivo bone formation capacity of three-dimensional 45S5 bioactive glass-based crystalline scaffolds.

Recent studies have demonstrated that surface characteristics, porosity, and mechanical strength of three-dimensional 45S5-type bioactive glass (BG)-based scaffolds are directly correlated with osteogenic properties. Three-dimensional BG-based scaffolds obtained from maritime natural sponges (MNSs) as sacrificial templates exhibit the required morphological properties; however, no in vivo data about the osteogenic features are available. In this study, uncoated (Group A) and gelatin-coated (Group B) crystalline MNS-obtained BG-based scaffolds were evaluated mechanically and seeded with human mesenchymal stem cells prior to subcutaneous implantation in immunodeficient mice. Before implantation and after explantation, micro-computed tomography scans were conducted, and scaffolds were finally subjected to histomorphometry. Scaffolds of both groups showed bone formation. However, Group B scaffolds performed distinctly better as indicated by a significant increase in scaffold volume (8.95%, p=0.039) over the implantation period compared with a nonsignificant increase of 5.26% in Group A scaffolds in micro-computed tomography analysis. Furthermore, percentage bone area was 10.33% (±1.18%) in the Group B scaffolds, which was significantly (p=0.007) higher compared with the 8.53% (±0.77%) in the Group A scaffolds in histomorphometry. Compressive strength was enhanced significantly by gelatin coating (9±2 vs. 4±1MPa; p=0.029). The presence of gelatin on the remnant parts was verified by scanning electron microscopy and X-ray spectroscopy, demonstrating the coatings' resilience. MNS-obtained BG-based scaffolds were thus confirmed to exhibit osteogenic properties in vivo that can significantly be enhanced by gelatin coating.

Mechanical behavior of a soft hydrogel reinforced with three-dimensional printed microfibre scaffolds

Reinforcing hydrogels with micro-fibre scaffolds obtained by a Melt-Electrospinning Writing (MEW) process has demonstrated great promise for developing tissue engineered (TE) constructs with mechanical properties compatible to native tissues. However, the mechanical performance and reinforcement mechanism of the micro-fibre reinforced hydrogels is not yet fully understood. In this study, FE models, implementing material properties measured experimentally, were used to explore the reinforcement mechanism of fibre-hydrogel composites. First, a continuum FE model based on idealized scaffold geometry was used to capture reinforcement effects related to the suppression of lateral gel expansion by the scaffold, while a second micro-FE model based on micro-CT images of the real construct geometry during compaction captured the effects of load transfer through the scaffold interconnections. Results demonstrate that the reinforcement mechanism at higher scaffold volume fractions was dominated by the load carrying-ability of the fibre scaffold interconnections, which was much higher than expected based on testing scaffolds alone because the hydrogel provides resistance against buckling of the scaffold. We propose that the theoretical understanding presented in this work will assist the design of more effective composite constructs with potential applications in a wide range of TE conditions.

Micro-Computed-Tomography-Guided Analysis of In Vitro Structural Modifications in Two Types of 45S5 Bioactive Glass Based Scaffolds.

Three-dimensional 45S5 bioactive glass (BG)-based scaffolds are being investigated for bone regeneration. Besides structural properties, controlled time-dependent alteration of scaffold morphology is crucial to achieve optimal scaffold characteristics for successful bone repair. There is no in vitro evidence concerning the dependence between structural characteristics and dissolution behavior of 45S5 BG-based scaffolds of different morphology. In this study, the dissolution behavior of scaffolds fabricated by the foam replica method using polyurethane foam (Group A) and maritime sponge Spongia Agaricina (Group B) as sacrificial templates was analyzed by micro-computed-tomography (µCT). The scaffolds were immersed in Dulbecco’s Modified Eagle Medium for 56 days under static cell culture conditions and underwent µCT-analysis initially, and after 7, 14, and 56 days. Group A showed high porosity (91%) and trabecular structure formed by macro-pores (average diameter 692 µm ± 72 µm). Group-B-scaffolds were less porous (51%), revealing an optimal pore size distribution within the window of 110–500 µm pore size diameter, combined with superior mechanical stability. Both groups showed similar structural alteration upon immersion. Surface area and scaffold volume increased whilst density decreased, reflecting initial dissolution followed by hydroxycarbonate-apatite-layer-formation on the scaffold surfaces. In vitro- and/or in vivo-testing of cell-seeded BG-scaffolds used in this study should be performed to evaluate the BG-scaffolds’ time-dependent osteogenic properties in relation to the measured in vitro structural changes.