Effect of In Vitro Culture Length on the Bone-Forming Capacity of Osteoblast-Derived Decellularized Extracellular Matrix Melt Electrowritten Scaffolds.

Recent advancements in decellularization have seen the development of extracellular matrix (ECM)-decorated scaffolds for bone regeneration; however, little is understood of the impact of in vitro culture prior to decellularization on the performances of these constructs. Therefore, this study investigated the effect of in vitro culture on ECM-decorated melt electrowritten polycaprolactone scaffold bioactivity. The scaffolds were seeded with osteoblasts and cultured for 1, 2, or 4 weeks to facilitate bone-specific ECM deposition and subsequently decellularized to form an acellular ECM-decorated scaffold. The utilization of mild chemicals and DNase was highly efficient in removing DNA while preserving ECM structure and composition. ECM decoration of the melt electrowritten fibers was observed within the first week of culture, with increased ECM at 2 and 4 week culture periods. Infiltration of re-seeded cells as well as overall bone regeneration in a rodent calvarial model was impeded by a longer culture period. Thus, it was demonstrated that the length of culture has a key influence on the osteogenic properties of decellularized ECM-decorated scaffolds, with long-term culture (2+ weeks) causing pore obstruction and creating a physical barrier which interfered with bone formation. These findings have important implications for the development of effective ECM-decorated scaffolds for bone regeneration.

3D printing for bone regeneration: challenges and opportunities for achieving predictability.

3D printing offers attractive opportunities for large-volume bone regeneration in the oro-dental and craniofacial regions. This is enabled by the development of CAD-CAM technologies that support the design and manufacturing of anatomically accurate meshes and scaffolds. This review describes the main 3D-printing technologies utilized for the fabrication of these patient-matched devices, and reports on their pre-clinical and clinical performance including the occurrence of complications for vertical bone augmentation and craniofacial applications. Furthermore, the regulatory pathway for approval of these devices is discussed, highlighting the main hurdles and obstacles. Finally, the review elaborates on a variety of strategies for increasing bone regeneration capacity and explores the future of 4D bioprinting and biodegradable metal 3D printing.

Tissue integration and biodegradation of soft tissue substitutes with and without compression: an experimental study in the rat.

OBJECTIVES: To analyze the influence of compression on tissue integration and degradation of soft tissue substitutes. MATERIAL AND METHODS: Six subcutaneous pouches in twenty-eight rats were prepared and boxes made of Al2O3 were implanted and used as carriers for soft tissue substitutes: a collagen matrix (MG), two volume-stable collagen matrices (FG/MGA), and a polycaprolactone scaffold(E). The volume-stable materials (FG/MGA/E) were further implanted with a twofold (2) and a fourfold (4) compression, created by the stacking of additional layers of the substitute materials. The samples were retrieved at 1, 2, and 12 weeks (10 groups, 3 time points, n = 5 per time point and group, overall, 150 samples). The area fraction of infiltrated fibroblasts and inflammatory cells was evaluated histologically. Due to within-subject comparisons, mixed models were conducted for the primary outcome. The level of significance was set at 5%. RESULTS: The area fraction of fibroblasts increased in all groups over time. At 12 weeks, the densely compressed materials FG4 (1.1%), MGA4 (1.7%), and MGA2 (2.5%) obtained lower values as compared to the other groups, ranging between 4.7 (E2) and 6.5% (MG). Statistically significant differences (p ≤ 0.05) were observed between groups FG4 vs MG/FG2/E/E4 as well as between MGA4 vs MG/FG2/E/E4 and E vs MGA2. CONCLUSIONS: Higher levels of compression led to delayed tissue integration. The effect of different compression levels was more distinct when compared to the differences between the materials. CLINICAL RELEVANCE: All biomaterials demonstrated tissue integration and a minimal concomitant inflammatory reaction. Clinically, it might be more favorable to obtain a sufficient flap release or to reduce the material size to improve the tissue integration processes.

Iron accumulation is associated with periodontal destruction in a mouse model of HFE-related haemochromatosis.

OBJECTIVE: To investigate the effect of Hfe gene mutation on the distribution of iron and periodontal bone loss in periodontal tissues. BACKGROUND DATA: It remains unclear how tissue iron loading affects the periodontium architectures in a genetic animal model of hereditary haemochromatosis (HH). METHODS: Male C57BL/6 Hfe -/- (8 weeks old) and wild-type (WT) mice were utilized to examine the iron distribution in periodontal tissues, as well as periodontal tissues changes using micro-computed tomography and histomorphometric analysis. Furthermore, tissue inflammatory mediators, bone markers and periodontal pathogens were carried out in PFA-fixed paraffin-embedded tissues using ELISA, RT-qPCR and genomic DNA qPCR, respectively. RESULTS: Excessive iron deposition was found in the periodontal ligament, gingiva and alveolar bone in Hfe -/- mice relative to their WT counterparts. This, in turn, was associated with significant periodontal bone loss, increased cemento-enamel junction-alveolar bone crest distance and decreased expression of molecules involved in bone development and turnover. Furthermore, the pro-inflammatory cytokine - interleukin 6 and periodontal bacteria - Campylobacter rectus were significantly increased in Hfe -/- mice compared with WT controls. CONCLUSION: Our results suggest that the iron loading in a mouse model of HH decreases alveolar bone formation and leads to alterations in the inflammatory state in the periodontium. Periodontal health should be assessed during the clinical assessment of HFE-HH patients.

Enamel matrix derivative promotes new bone formation in xenograft assisted maxillary anterior ridge preservation-A randomized controlled clinical trial.

OBJECTIVES: To compare the effectiveness of deproteinized bovine bone mineral with 10% collagen alone (DBBMC) or with enamel matrix derivative (DBBMC-EMD) in ridge preservation. METHODS: 42 maxillary anterior teeth were extracted and received either a DBBMC (control) or DBBMC-EMD (test) treatment protocol. CBCT taken before and 4 months after the extraction procedure was used to measure changes in alveolar ridge width (RW), buccal bone height (BH) and palatal bone height (PH). Bone cores were harvested during implant osteotomy preparation, and the samples processed histomorphometrically to assess the fraction of new bone (%NB), residual graft (%RG) and soft tissue matrix (%STM). RESULTS: Overall, both treatment groups showed significant reductions in mean RW from baseline to 4 months after extraction, but no significant change in either mean BH or PH over this time. When CBCT measurements were analysed according to the initial thickness of the buccal wall (BT < 1 mm vs. BT ≥ 1 mm), significant reductions in all ridge dimensions (RW, BH and PH) were noted in the <1 mm BT group. Histomorphometrically, the DBBMC-EMD test group showed significantly increased new bone formation (%NB): (control = 16.5 ± 6.9% cf.; test = 45.1 ± 8.8%) with less residual graft (%RG): (control = 36.8 ± 8.8% cf.; test = 20.3 ± 7.2%) compared to the DBBMC control group. CONCLUSIONS: Both DBBMC alone and DBBMC-EMD treated sites 4 months after extraction lost RW but showed no significant change in BH or PH. Irrespective of treatment, maxillary anterior teeth with thick initial buccal walls (≥1 mm) exhibited less alveolar ridge reduction 4 months after treatment. The addition of EMD to DBBMC resulted in more new bone formation in the test group.

Fibre guiding scaffolds for periodontal tissue engineering.

The periodontium is a highly hierarchically organized organ composed of gingiva, alveolar bone, periodontal ligament and cementum. Periodontitis leads to the destruction of hard and soft tissues ultimately leading to a loss of the teeth supporting apparatus. Current treatments are capable of limiting the disease progression; however, true regeneration, characterized by perpendicularly oriented periodontal ligament fibre attachment to cementum on the root surface remains challenging. Tissue engineering approaches have been developed to enhance regeneration via micro-engineered topographical features, purposely designed to guide the insertion of the regenerated ligament to the root surface. This review reports on the recent advancements in scaffold manufacturing methodologies for generating fibre guiding properties and provides a critical insight in the current limitations of these techniques for the formation of functional periodontal attachment.

Workflow for highly porous resorbable custom 3D printed scaffolds using medical grade polymer for large volume alveolar bone regeneration.

OBJECTIVES: This study investigates the design, workflow, and manufacture of highly porous, resorbable additively manufactured, 3-dimensional (3D) custom scaffolds for the regeneration of large volume alveolar bone defects. MATERIALS AND METHODS: Computed tomography (CT) scans of 5 posterior mandibular vertical bone defects were obtained. Surface masks (3D surface contours) of the recipient site were first isolated using a contrast threshold, transformed into 3D objects, and used to guide the formation of custom implant template models. To determine model accuracy and fit, the gap and overlap between the patient geometry models and the idealized template 3D models were quantified. Models were 3D printed from medical grade polycaprolactone (PCL) into porous scaffolds. For scaffold dimensional quantification, scaffolds were scanned using a micro-computed tomography (µCT) scanner. RESULTS: The design and printing processes each achieved dimensional errors of <200 µm on average. The average gap between the template implant model and the scanned scaffold model was found to be 74 ± 14 µm. The printed scaffold was confirmed as having a porosity of 83.91%, a mean polymer or filament thickness of 200 ± 46 µm, and a mean pore size of 590 ± 243 µm. CONCLUSION: The approach described in this study is straightforward, adaptable to a range of patient geometries, and results in the formation of reproducible, dimensionally accurate custom implants. These highly porous 3D structures manufactured from resorbable medical grade material represent a potentially transformative technology toward the clinical implementation of scaffold-guided bone regeneration procedures.

The influence of high-dose systemic zoledronate administration on osseointegration of implants with different surface topography.

AIM: To evaluate the influence of systemic zoledronate administration on the osseointegration of titanium implants with different surface topography in rat maxillae. METHODS: Twenty Sprague-Dawley rats were divided into two groups-test (bisphosphonate) and control (healthy). Bisphosphonate administration began three weeks prior to implant placement, and the animals received zoledronate (66 µg/kg) three times per week. Forty endosseous implants with a moderately rough (20 implants) or a turned surface (20 implants) were immediately placed bilaterally into extraction sockets of maxillary first molars. Animals were sacrificed after 14 and 28 days of healing, and en bloc specimens were harvested for histological and histomorphometric analysis. Osseointegration was quantified by measuring the percentage of bone-to-implant contact. RESULTS: Bone-to-implant contact (BIC) (mean ± SD) values of moderately rough and turned implants at day 14 in test group were 17.62 ± 6.68 and 10.69 ± 1.48, respectively, while in the control group, they were 46.36 ± 5.08 and 33.29 ± 8.89, respectively. At day 28, BIC values of moderately rough and turned implants in the test group were 25.94 ± 7.87 and 7.83 ± 4.30, respectively, while in the control group, they were 72.99 ± 6.60 and 47.62 ± 18.19, respectively. Statistically significant higher BIC values were measured on moderately rough implants compared to turned implants at 28 days, and the control group compared to the test group for both implant surfaces. Histological observations for the control and the test groups demonstrated initial bone formation around moderately rough implants not only on the surface of the parent bone, as was the case with the turned surfaced implants, but also along the implant surface itself. CONCLUSIONS: Systemic zoledronate administration negatively influences osseointegration. Osseointegration was enhanced adjacent to moderately rough compared to turned implants in both the presence and absence of systemic zoledronate administration. Therefore, topographical surface modification may partially offset the negative impact of zoledronate administration.

Systematic Comparison of the Effect of Four Clinical-Grade Platelet Rich Hemoderivatives on Osteoblast Behaviour.

Hemoderivatives have utilized in an empirical manner, driven by clinical considerations, leading to the development of a plethora of manufacturing protocols. The purpose of this study was to investigate the composition and bioactivity of four common clinical-grade hemoderivates prepared using standardised methods. Four different hemoderivatives were obtained from sheep blood and divided into two groups: A-PRF/i-PRF (fresh) and P-PRP/L-PRP (anticoagulated). Thrombus (CLOT) was used as a control. Thrombocyte quantification, growth factor composition (IGF-I, VEGF, PDGF-BB, BMP-2), cell viability, migration and mineralization assay were evaluated. Platelet recovery was superior for L-PRP followed by P-PRP. A significant cumulative release of IGF-I and PDGF-BB was noted for A-PRF and L-PRP groups at early time points. Similar release profiles of BMP-2 and VEGF were noted in all protocols. Cell viability and migration assay have demonstrated a detrimental effect when the concentration was ≥60%. Moreover, at Day 21, i-PRF have demonstrated superior mineralisation properties when compared to all groups. A negative impact of A-PRF was demonstrated at high concentrations. Despite its low content in growth factors, i-PRF was the best performing blood product for inducing osteoblast mineralisation, and therefore could be the candidate of choice for utilisation in bone tissue engineering applications.

The effect of decellularized tissue engineered constructs on periodontal regeneration.

AIM: To evaluate the effect of decellularized tissue engineered constructs on cell differentiation in vitro and periodontal regeneration in vivo. MATERIALS AND METHODS: Periodontal ligament cell (PDLC) sheets were loaded on polycaprolactone (PCL) scaffolds and then decellularized. Constructs were assessed for their effect on allogenic PDLC and mesenchymal stem cell (MSC) differentiation in vitro, as evaluated by gene expression of bone and periodontal ligament tissue markers post-seeding. Expression of MSC marker STRO-1 was assessed by immunostaining. Decellularized constructs were evaluated in a rat periodontal defect model to assess their biocompatibility and tissue integration. Microcomputed topography (µCT) and histological assessment were performed to assess the regenerative potential of the constructs at 2 and 4 weeks postoperatively. RESULTS: There was upregulation of bone marker gene expression by PDLCs especially on the 14th day. MSCs lacked bone markers expression, but showed increased collagen I marker expression on day 14. STRO-1 expression by the MSCs decreased over the three timepoints when seeded on decellularized sheets. Histological assessment demonstrated the biocompatibility of the decellularized constructs in vivo. More new attachment formation was observed on the decellularized constructs compared to scaffold only controls. CONCLUSION: Decellularized tissue engineered constructs are capable of inducing cell differentiation in vitro and have the potential to facilitate periodontal regeneration in vivo.

3-Dimensional functionalized polycaprolactone-hyaluronic acid hydrogel constructs for bone tissue engineering.

AIM: Alveolar bone regeneration remains a significant clinical challenge in periodontology and dental implantology. This study assessed the mineralized tissue forming potential of 3-D printed medical grade polycaprolactone (mPCL) constructs containing osteoblasts (OB) encapsulated in a hyaluronic acid (HA)-hydrogel incorporating bone morphogenetic protein-7 (BMP-7). MATERIALS AND METHODS: HA-hydrogels containing human OB ± BMP-7 were prepared. Cell viability, osteogenic gene expression, mineralized tissue formation and BMP-7 release in vitro, were assessed by fluorescence staining, RT-PCR, histological/µ-CT examination and ELISA respectively. In an athymic rat model, subcutaneous ectopic mineralized tissue formation in mPCL-hydrogel constructs was assessed by µ-CT and histology. RESULTS: Osteoblast encapsulation in HA-hydrogels did not detrimentally effect cell viability, and 3-D culture in osteogenic media showed mineralized collagenous matrix formation after 6 weeks. BMP-7 release from the hydrogel was biphasic, sustained and increased osteogenic gene expression in vitro. After 4 weeks in vivo, mPCL-hydrogel constructs containing BMP-7 formed significantly more volume (mm3 ) of vascularized bone-like tissue. CONCLUSIONS: Functionalized mPCL-HA hydrogel constructs provide a favourable environment for bone tissue engineering. Although encapsulated cells contributed to mineralized tissue formation within the hydrogel in vitro and in vivo, their addition did not result in an improved outcome compared to BMP-7 alone.

Comparison of early osseointegration of SLA® and SLActive® implants in maxillary sinus augmentation: a pilot study.

OBJECTIVE: To assess the impact of a hydrophilic implant surface (SLActive® ) placed into augmented maxillary sinuses on bone-to-implant contact (BIC) and surrounding tissue composition when compared to a hydrophobic surface (SLA® ). MATERIAL AND METHODS: Four sheep underwent bilateral sinus augmentation. Each sinus received anorganic bovine bone mineral + autogenous bone (ABBM + AB). Sixteen implants were subsequently placed 12 weeks postgrafting with each sinus receiving a control (SLA® ) and test implant (SLActive® ). Two animals were sacrificed at 2 weeks and another two animals were sacrificed at 4 weeks postimplantation. The eight sinuses and 16 implants were processed for histomorphometry, which assessed bone-to-implant contact (%BIC) and tissue elements (woven bone - WB, lamellar bone - LB, soft tissue - ST) in the interthread region of implants within the augmented sinus. RESULTS: There was a statistically significant increase in %BIC at week 4 compared to the week 2 animals in both test (P < 0.005) and control (P < 0.005) groups. There was a statistically significant greater %BIC around test implants when compared to control implants in both week 2 (P < 0.05) and week 4 animals (P < 0.05). Greater %WB (11.17% ±6.82) and %LB (11.06% ±3.67) were seen in the test implants when compared to the control implants independent of time. This was only statistically significant for %LB (P < 0.05). A statistically significant reduction of 16.78% (±6.19) in %ST was noted in test implants when compared to control implants (P < 0.05) independent of time. CONCLUSION: Both time and the use of hydrophilic implant surface had a positive impact on %BIC around implants placed into augmented maxillary sinuses. Hydrophilic implant surfaces also had a positive impact on surrounding tissue composition. Larger trials are needed to better assess and detect differences between these two surfaces in augmented maxillary sinuses.

Tissue Engineering in Hand Surgery: A Technology Update.

The field of hand surgery is constantly evolving to meet the challenges of repairing intricate anatomical structures with limited availability of donor tissue. The past 10 years have seen an exponential growth in tissue engineering, which has broadened the perspectives of tackling these age-old problems. Various fabrication techniques such as melt electrospinning and fused deposition modelling have been employed to synthesize 3-dimensional bioscaffolds that can be used to replace lost tissue. These bioscaffolds with strategic biomimicry have been shown to allow for integrative and functional repair of tissue injuries. This review article summarizes the most current advances in tissue engineering and its applications in the field of hand surgery. It outlines the current tissue engineering techniques commonly used for tackling musculoskeletal problems and highlights the most promising approaches according to clinical evidence. In particular, the paper explores regenerative medicine concepts applied to specific tissues including nerve, bone, cartilage, tendon, ligament, and vessels. In the face of innovative and pioneering research, tissue engineering will undoubtedly play a key role in reconstructive hand surgery in the not too distant future.

Neurological heterotopic ossification following spinal cord injury is triggered by macrophage-mediated inflammation in muscle.

Neurological heterotopic ossification (NHO) is the abnormal formation of bone in soft tissues as a consequence of spinal cord or traumatic brain injury. NHO causes pain, ankyloses, vascular and nerve compression and delays rehabilitation in this high-morbidity patient group. The pathological mechanisms leading to NHO remain unknown and consequently there are no therapeutic options to prevent or reduce NHO. Genetically modified mouse models of rare genetic forms of heterotopic ossification (HO) exist, but their relevance to NHO is questionable. Consequently, we developed the first model of spinal cord injury (SCI)-induced NHO in genetically unmodified mice. Formation of NHO, measured by micro-computed tomography, required the combination of both SCI and localized muscular inflammation. Our NHO model faithfully reproduced many clinical features of NHO in SCI patients and both human and mouse NHO tissues contained macrophages. Muscle-derived mesenchymal progenitors underwent osteoblast differentiation in vitro in response to serum from NHO mice without additional exogenous osteogenic stimuli. Substance P was identified as a candidate NHO systemic neuropeptide, as it was significantly elevated in the serum of NHO patients. However, antagonism of substance P receptor in our NHO model only modestly reduced the volume of NHO. In contrast, ablation of phagocytic macrophages with clodronate-loaded liposomes reduced the size of NHO by 90%, supporting the conclusion that NHO is highly dependent on inflammation and phagocytic macrophages in soft tissues. Overall, we have developed the first clinically relevant model of NHO and demonstrated that a combined insult of neurological injury and soft tissue inflammation drives NHO pathophysiology.

A histomorphometric assessment of collagen-stabilized anorganic bovine bone mineral in maxillary sinus augmentation - a prospective clinical trial.

OBJECTIVE: To histomorphometrically compare the use of collagen-stabilized anorganic bovine bone (ABBM-C) (test) to anorganic bovine bone + autogenous bone (ABBM + AB) (control) in maxillary sinus augmentation. MATERIALS AND METHODS: Forty (n = 40 sinuses) patients underwent sinus augmentation and received either control (20 sinuses) or test bone graft (20 sinuses). Bone samples were harvested from the augmented sinuses 5 months postgrafting. The samples were processed for histomorphometry, which assessed within the primary region of interest (ROI-1), the area fraction of new bone (%NB), graft particle osseointegration (% OI), residual graft (%RG), and soft tissue components (% STM). The same analysis was also carried out in a second region of interest (ROI-2) located in a zone 1 mm proximal to the previous maxillary sinus floor. RESULTS: In both ROI-1 and ROI-2, the mean % NB, %RG, and %STM in the control group were similar to mean values in the test group. The % OI was significantly greater in the control group (42.0 +/- 26.8) when compared to the test group (19.6 +/- 27.3) in ROI-2 (P < 0.05). No statistically significant differences were seen when ROI-1 and ROI-2 were compared except for improved %OI in ROI-2 in the control group. The mean proportion of lamellar bone to woven bone in the control group (1.22 ± 1.48) was significantly greater than the test group (0.38 ± 0.29) (P < 0.05). CONCLUSION: ABBM-C exhibited very similar histomorphometric parameters to the composite graft of ABBM + AB. The ABBM + AB group was more mature as indicated by the significantly greater proportion of lamellar bone when compared to the ABBM-C. Improved % OI was seen in the zone proximal to the resident bony floor in the ABBM + AB group. Based on histological assessment, ABBM-C is a suitable bone substitute for the purposes of maxillary sinus augmentation. Its clinical utility may be indicated in cases of sinus membrane perforation and insufficient autogenous bone in the local area.

A histomorphometric assessment of collagen-stabilized anorganic bovine bone mineral in maxillary sinus augmentation - a randomized controlled trial in sheep.

OBJECTIVE: To histomorphometrically compare the use of collagen-stabilized anorganic bovine bone (ABBM-C) (test) to anorganic bovine bone + autogenous bone (ABBM + AB) (control) in maxillary sinus augmentation. MATERIALS AND METHODS: Nine sheep underwent bilateral sinus augmentation. Each sinus was randomized to receive either control or test bone graft. Three animals were sacrificed at 8 weeks, and six animals were sacrificed at 16 weeks post-grafting. The 18 sinuses were processed for histomorphometry, which assessed the area fraction of new bone (%NB), residual graft (%RG) and soft tissue components (% STM), as well as graft particle osseointegration (% OI), within three zones equally distributed from the augmented sinus floor. RESULTS: At week 16, a significant increase in %NB was evident across all three zones in the control group when compared to week 8. A significantly greater %NB was evident in the control group when compared to the test group in zones 2 (P < 0.001) and 3 (P < 0.001). There was a significant increase in %OI in week 16 when compared to week 8 across all three zones in the control group (P < 0.001). %OI in the control group was significantly greater across all three zones when compared to the test group at week 16 (P < 0.001). Zone was found to be a significant main effect (P < 0.001) that was independent of time and treatment with decreasing %OI in distant zones. %RG did not significantly change with time for both groups. There was a significant reduction in %ST in week 16 when compared to week 8 across all three zones in the control group (P < 0.001). %ST in the test group was significantly greater across all zones when compared to the control group at week 16 (P < 0.001). CONCLUSION: Both groups exhibited very similar histomorphometric measurements in the zones proximal to the resident sinus wall. The % NB and % OI were greatest in the zones proximal to resident bony walls and gradually decreased as the distance from the proximal walls increased. There was greater % NB and % OI in the control group when compared to the test group in the distant zone.

Advanced tissue engineering scaffold design for regeneration of the complex hierarchical periodontal structure.

AIM: This study investigated the ability of an osteoconductive biphasic scaffold to simultaneously regenerate alveolar bone, periodontal ligament and cementum. MATERIALS AND METHODS: A biphasic scaffold was built by attaching a fused deposition modelled bone compartment to a melt electrospun periodontal compartment. The bone compartment was coated with a calcium phosphate (CaP) layer for increasing osteoconductivity, seeded with osteoblasts and cultured in vitro for 6 weeks. The resulting constructs were then complemented with the placement of PDL cell sheets on the periodontal compartment, attached to a dentin block and subcutaneously implanted into athymic rats for 8 weeks. Scanning electron microscopy, X-ray diffraction, alkaline phosphatase and DNA content quantification, confocal laser microscopy, micro computerized tomography and histological analysis were employed to evaluate the scaffold's performance. RESULTS: The in vitro study showed that alkaline phosphatase activity was significantly increased in the CaP-coated samples and they also displayed enhanced mineralization. In the in vivo study, significantly more bone formation was observed in the coated scaffolds. Histological analysis revealed that the large pore size of the periodontal compartment permitted vascularization of the cell sheets, and periodontal attachment was achieved at the dentin interface. CONCLUSIONS: This work demonstrates that the combination of cell sheet technology together with an osteoconductive biphasic scaffold could be utilized to address the limitations of current periodontal regeneration techniques.

Melt electrowriting scaffolds with fibre-guiding features for periodontal attachment.

Periodontal regeneration requires the re-attachment of oblique and perpendicular periodontal ligament (PDL) fibres to newly formed cementum and alveolar bone, which has proven elusive with existing approaches. In this study, multiple fibre-guiding biphasic tissue engineered constructs were fabricated by melt electrowriting. The biphasic scaffolds were 95 % porous and consisted of a pore size gradient bone compartment and periodontal compartment made of fibre-guiding channels with micro-architectural features ranging from 100 to 60 µm aimed to direct PDL fibre alignment and attachment. In vitro evaluations over 3 and 7 days demonstrated a marked improvement in collagen fibre orientation (over 60 % fully aligned) for scaffolds with micro-architecture ≤100 µm. The biphasic scaffolds were placed on a dentine slice and implanted ectopically, and this demonstrated that all micro-channels groups facilitated oblique and perpendicular alignment and attachment on the dentine with a mean nuclei angle and mean collagen fibre angle of approximately 60° resembling the native periodontal ligament attachment. A further in vivo testing using a surgically created rodent periodontal model highlighted the 80 µm micro-channel group's effectiveness, showing a significant increase in oblique PDL fibre attachment (72 %) and periodontal regeneration (56 %) when compared to all other groups onto the tooth root compared to control groups. Further to this, immunohistochemistry demonstrated the presence of periostin in the newly formed ligament indicating that functional regeneration occurred These findings suggest that scaffold micro-architectures of 100 µm or below can play a crucial role in directing periodontal tissue regeneration, potentially addressing a critical gap in periodontal therapy. STATEMENT OF SIGNIFICANCE: Periodontal regeneration remains a significant clinical challenge. Essential to restoring dental health and function is the proper attachment of the periodontal ligament, which is functionally oriented, to regenerated bone and cementum. Our research presents an innovative biphasic scaffold, utilizing Melt Electrowriting to systematically guide tissue growth. Distinct from existing methods, our scaffold is highly porous, adaptable, and precisely guides periodontal ligament fibre attachment to the opposing tooth root and alveolar bone interfaces, a critical step for achieving periodontal functional regeneration. Our findings not only bridge a significant gap in biomaterial driven tissue guidance but also promise more predictable outcomes for patients, marking a transformative advancement in the field.

Cell Seeding on 3D Scaffolds for Tissue Engineering and Disease Modeling Applications.

Scaffold cell seeding is a crucial step for the standardization and homogeneous maturation of tissue engineered constructs. This is particularly critical in the context of additively manufactured scaffolds whereby large pore size and high porosity usually impedes the retention of the seeding solution resulting in poor seeding efficacy and heterogeneous cell distribution. To circumvent this limitation, a simple yet efficient cell seeding technique is described in this chapter consisting of preincubating the scaffold in 100% serum for 1 h leading to reproducible seeding. A proof of concept is demonstrated using highly porous melt electrowritten polycaprolactone scaffolds as the cell carrier. As cell density, cell distribution, and differentiation within the scaffold are important parameters, various assays are proposed to validate the seeding and perform quality control of the cellularized construct using techniques such as alizarin red, Sirius red, and immunostaining.

The effect of culture conditions on the bone regeneration potential of osteoblast-laden 3D bioprinted constructs.

Three Dimensional (3D) bioprinting is one of the most recent additive manufacturing technologies and enables the direct incorporation of cells within a highly porous 3D-bioprinted construct. While the field has mainly focused on developing methods for enhancing printing resolution and shape fidelity, little is understood about the biological impact of bioprinting on cells. To address this shortcoming, this study investigated the in vitro and in vivo response of human osteoblasts subsequent to bioprinting using gelatin methacryloyl (GelMA) as the hydrogel precursor. First, bioprinted and two-dimensional (2D) cultured osteoblasts were compared, demonstrating that the 3D microenvironment from bioprinting enhanced bone-related gene expression. Second, differentiation regimens of 2-week osteogenic pre-induction in 2D before bioprinting and/or 3-week post-printing osteogenic differentiation were assessed for their capacity to increase the bioprinted construct's biofunctionality towards bone regeneration. The combination of pre-and post-induction regimens showed superior osteogenic gene expression and mineralisation in vitro. Moreover, a rat calvarial model using microtomography and histology demonstrated bone regeneration potential for the pre-and post-differentiation procedure. This study shows the positive impact of bioprinting on cells for osteogenic differentiation and the increased in vivo osteogenic potential of bioprinted constructs via a pre-induction method. STATEMENT OF SIGNIFICANCE: 3D bioprinting, one of the most recent technologies for tissue engineering has mostly focussed on developing methods for enhancing printing properties, little is understood on the biological impact of bioprinting and /or subsequent in vitro maturation methods on cells. Therefore, we addressed these fundamental questions by investigating osteoblast gene expression in bioprinted construct and assessed the efficacy of several induction regimen towards osteogenic differentiation in vitro and in vivo. Osteogenic induction of cells prior to seeding in scaffolds used in conventional tissue engineering applications has been demonstrated to increase the osteogenic potential of the resulting construct. However, to the best of our knowledge, pre-induction methods have not been investigated in 3D bioprinting.