Aim: To investigate the vascularization capacity of a decellularized dental pulp matrix (DDP) of bovine origin seeded with human dental pulp stem cells (hDPSCs) in vitro and to present preliminary in vivo findings.Methodology: Bovine dental pulps were decellularized and then analysed using histological staining and DNA quantification. The resultant DDPs were characterized using immunohistochemical staining for the retention of vascular endothelial growth factor A (VEGF‐A) and fibroblast growth factor 2 (FGF‐2). Furthermore, DDPs were recellularized with hDPSCs and analysed histologically. The expression of markers involved in angiogenesis by hDPSCs colonizing the DDPs was assessed in vitro. A preliminary in vivo study was then conducted in which hDPSCs‐seeded and unseeded DDPs were inserted in debrided human premolars root slices and implanted subcutaneously in immunodeficient mice. Samples were retrieved after 30 days and analysed using histological and immunohistochemical staining. The independent samples t‐test, analysis of variance and a Kruskal–Wallis test were used to analyse the quantitative data statistically depending on the group numbers and normality of data distribution. The difference between the groups was considered significant when the P‐value was less than 0.05.Results: Acellular dental pulp matrices were generated following bovine dental pulp decellularization. Evaluation of the developed DDPs revealed a significant DNA reduction (P < 0.0001) with preservation of the native histoarchitecture and vasculature and retention of VEGF‐A and FGF‐2. Upon recellularization of the DDPs with hDPSCs, the in vitro analyses revealed cell engraftment with progressive repopulation of DDPs’ matrices and vasculature and with enhanced expression of markers involved in angiogenesis. In vivo implantation of root slices with hDPSCs‐seeded DDPs revealed apparent vascularization enhancement as compared to the unseeded DDP group (P < 0.0001).Conclusions: The developed decellularized dental pulp matrix had pro‐angiogenic properties characterized by the retention of native vasculature and angiogenic growth factors. Seeding of hDPSCs into the DDP led to progressive repopulation of the vasculature, enhanced expression of markers involved in angiogenesis in hDPSCs and improved in vivo vascularization capacity. The se suggest that a combination of DDP and hDPSCs have the potential to provide a promising vascularization promoting strategy for dental pulp regeneration.

Chitosan (CS), a biopolymer, holds significant potential in bone regeneration due to its biocompatibility and biodegradability attributes. While crustacean-derived CS is conventionally used in research, there is growing interest in fungal-derived CS for its equally potent properties in bone regenerative applications. Here, we investigated the physicochemical and biological characteristics of fungal (MDC) and crustacean (ADC)-derived CS scaffolds embedded with different concentrations of tricalcium phosphate minerals (TCP), i.e., 0(wt)%: ADC/MDC-1, 10(wt)%: ADC/MDC-2, 20(wt)%: ADC/MDC-3 and 30(wt)%: ADC/MDC-4. ADC-1 and MDC-1 lyophilised scaffolds lacking TCP minerals presented the highest zeta potentials of 47.3 ± 1.2 mV and 55.1 ± 1.6 mV, respectively. Scanning electron microscopy revealed prominent distinctions whereby MDC scaffolds exhibited striation-like structural microarchitecture in contrast to the porous morphology exhibited by ADC scaffold types. With regard to the 4-week scaffold mass reductions, MDC-1, MDC-2, MDC-3, and MDC-4 indicated declines of 55.98 ± 4.2%, 40.16 ± 3.6%, 27.05 ± 4.7%, and 19.16 ± 5.3%, respectively. Conversely, ADC-1, ADC-2, ADC-3, and ADC-4 presented mass reductions of 35.78 ± 5.1%, 25.19 ± 4.2%, 20.23 ± 6.3%, and 13.68 ± 5.4%, respectively. The biological performance of the scaffolds was assessed through in vitro bone marrow mesenchymal stromal cell (BMMSCs) attachment via indirect and direct cytotoxicity studies, where all scaffold types presented no cytotoxic behaviours. MDC scaffolds indicated results comparable to ADC, where both CS types exhibited similar physiochemical properties. Our data suggest that MDC scaffolds could be a potent alternative to ADC-derived scaffolds for bone regeneration applications, particularly for 10(wt)% TCP concentrations.

Dicalcium Phosphate Dihydrate (DCPD) mineral scaffolds alone do not possess the mechanical flexibility, ease of physicochemical properties’ tuneability or suitable porosity required for regenerative bone scaffolds. Herein, we fabricated highly porous freeze-dried chitosan scaffolds embedded with different concentrations of Dicalcium Phosphate Dihydrate (DCPD) minerals, i.e., 0, 20, 30, 40 and 50 (wt)%. Increasing DCPD mineral concentration led to increased scaffold crystallinity, where the % crystallinity for CH, 20, 30, 40, and 50-DCPD scaffolds was determined to be 0.1, 20.6, 29.4, 38.8 and 69.9%, respectively. Reduction in scaffold pore size distributions was observed with increasing DCPD concentrations of 0 to 40 (wt)%; coalescence and close-ended pore formation were observed for 50-DCPD scaffolds. 50-DCPD scaffolds presented five times greater mechanical strength than the DCPD mineral-free scaffolds (CH). DCPD mineral enhanced cell proliferation for the 20, 30 and 40-DCPD scaffolds. 50-DCPD scaffolds presented reduced pore interconnectivity due to the coalescence of many pores in addition to the creation of closed-ended pores, which were found to hinder osteoblast cell proliferation.

Biomorphic hydroxyapatite scaffolds derived from rattan wood (GreenBone) show significant promise in bone tissue engineering due to their inherent structural similarity to natural bone. Laser-drilled GreenBone scaffolds were studied for enhanced porosity, nutrient diffusion, cellular infiltration, and vascularisation. Patient-derived bone marrow mesenchymal stromal/stem cells (BMMSCs) and culture-expanded mesenchymal stem cells (cMSCs) demonstrated high cell viability (>90%), considerable adhesion, and extensive cytoskeletal organisation. Trilineage differentiation confirmed the multipotency of BMMSCs, with osteogenic, adipogenic, and chondrogenic markers being successfully expressed. BMMSCs and cMSCs exhibited enhanced differentiation and gene expression profiles. At week 4, key osteogenic and angiogenic genes such as BMP2, VEGFC, RUNX2, and COL1A1 showed elevated expression, indicating improved bone formation and vascularisation activity. Markers associated with extracellular matrix (ECM) remodelling, including MMP9 and TIMP1, were also upregulated, suggesting active tissue remodelling. ELISA analysis for VEGF further demonstrated increased VEGF secretion, highlighting the scaffold’s angiogenic potential. The improved cellular response and vascular signalling emphasise the translational relevance of laser-modified GreenBone scaffolds for bone tissue engineering, particularly for critical-sized defect repair requiring rapid vascularised bone regeneration.