Poly-lactic acid (PLA) scaffolds are widely used in bone tissue engineering. The introduction of 3D printing has greatly increased the ability for tailoring different geometrical designs of these scaffolds for improved cellular attachment, growth and differentiation. This study aimed to investigate the effect of PLA fibre angle in 3D printed PLA scaffolds on hDPSC attachment and growth in vitro.

Two types of PLA scaffolds were prepared via 3D printing containing fibres angled at either 45° or 90°. hDPSCs (P4, 2*10⁵ cells per scaffold) were statically seeded for 4 hours on to the scaffolds (7×3.5×3 mm³, n=3). Cellular attachment was checked using fluorescence microscopy and the number of unattached cells was counted using a haemocytometer (HCM). The cell-scaffold constructs were then cultured in osteogenic medium for up to 5 weeks. ALP staining and SEM were performed for one construct from each group at week 3. Cellular viability was determined using CMFDA/EHD1 live/dead labelling at week 4. After 5 weeks, constructs were processed for histology.

Fluorescence micrographs showed high numbers of hDPSCs attached to scaffold surfaces in both groups after seeding irrespective of fibre angle. However, HCM cell count revealed that the 45° angled PLA scaffolds had significantly greater cell attachment compared to the 90° angled PLA group (p<0.0001). After 3 weeks in osteogenic culture, both types of construct showed strong ALP staining. SEM showed that in the 45° angled PLA group, almost all macro-pores were fully closed with newly formed cell sheets. In comparison, in the 90° angled group, most of the macro-pores remained open although a limited amount of cellular bridging was present. SEM also detected crystal deposits in different areas within the cell sheets for both construct groups. Most hDPSCs were alive in both groups at week 4 of culture with few dead cells present. After 5 weeks, histology showed marked cellular growth and new matrix formation, with detectable Van Kossa +ve crystal deposits in different areas within all constructs irrespective of PLA fibre angle.

This study showed that 45° angled PLA 3D printed scaffolds enhanced hDPSC attachment and cellular bridging, which may help to rapidly close the macro-pores within the scaffold compared to the 90° angled group. This illustrates the potential of 45° angled 3D printed PLA scaffolds as good candidates for bone tissue engineering.

Stromal cells derived from human dental pulp (HDPSCs) are of current interest for applications in skeletal tissue engineering. Angiogenesis and revascularization of bone grafts or bone constructs in vivo are of paramount importance for bone tissue regeneration and/or fracture healing. The aim of this study was to investigate the angiogenic and osteogenic potential of HDPSCs in combination with Bioglass¯ scaffolds in vitro and in vivo.

HDPSCs, isolated by collagenase digestion, were either maintained as monolayers or dynamically seeded on 3D Bioglass¯ scaffolds and cultured under either basal or osteogenic conditions for 2 and 4 weeks. Expression of osteogenic (COL1A1, ALP, RUNX2 and OC) and angiogenic markers (VEGFR2, CD34 and PECAM1) was determined using qRT-PCR. Alternatively, constructs were either cultured in vitro under basal/osteogenic conditions for 6 weeks or sealed in diffusion chambers which were then implanted intraperitoneally in immunosuppressed mice for 8 weeks. Retrieved constructs were fixed and embedded for histology and immunohistochemistry using antibodies against COL1, RUNX2, OC, VEGFR2, CD34 and PECAM1. qRT-PCR showed no significant differences in gene expression of osteogenic markers between basal and osteogenic media for both 3D construct and monolayers.

However when comparing 3D constructs to monolayers: COL1A1 showed a significantly lower expression (p< 0.05) in 3D compared to 2D at 2 weeks in both culture conditions, and this pattern was reversed after 4 weeks. ALPL was significantly lower in 3D constructs at 2 weeks under both conditions (p<0.01), and was significantly higher in basal conditions at 4 weeks (p<0.05). RUNX2 showed higher expression in 3D constructs at all time points and under both conditions while OC showed lower expression in 3D constructs at 2 weeks and higher expression at 4 weeks under both conditions. For the angiogenic markers, 3D constructs under osteogenic conditions showed an increase of expression in VEGFR2 and PECAM1 at 2 weeks followed by a decrease at week 4, while CD34 expression was undetected in 3D constructs at all times and under both sets of culture conditions. The expression of VEGFR2 and PECAM 1 under both conditions and at both time points was greater in 3D constructs compared to monolayers. After 8 weeks, the in vivo retrieved constructs showed no signs of inflammatory reactions. Immunohistology confirmed positive staining of osteogenic and angiogenic markers in 3D constructs from both in vitro and in vivo experiments with a greater staining intensity seen in the in vivo constructs. Furthermore, the in vivo constructs showed more intense sirius red staining and higher intensity of immunostaining using antibodies to type 1 collagen, with higher calcification as indicated by alizarin red staining.

In conclusion, this study indicated that a combination of HDPSCs and Bioglass¯ scaffolds has potential to provide a suitable microenvironment for angiogenic and osteogenic differentiation of HDPSCs which is essential for bone regeneration in preclinical and/or clinical applications.

Introduction

P-15 (GTPGPQGIAGQRGVV), a fifteen residue synthetic peptide, is a structural analogue of the cell binding domain of Type 1 collagen and creates a biomimetic environment for bone repair when immobilized on anorganic bovine mineral (ABM) scaffolds. ABM-P-15 scaffolds have been shown to enhance bone marrow stromal cell growth and differentiation. This study aimed at evaluating the osteogenic potential of human dental pulp stromal cells (HDPSCs) compared to human bone marrow stromal cells (HBMSCs) in monolayer and on 3D ABM-P-15 scaffolds in vitro and in vivo.

Introduction: Articular cartilage has limited capacity for regeneration. Tissue engineering strategies offer future hope for cartilage replacement and repair. In an attempt to mimic functional native cartilage for tissue repair, current research focuses on construct/implant designs that simulate an embryonic like microenvironment to promote cellular differentiation along a chondrogenic lineage. The aim of the present study was, for the first time, to illustrate the differences between human neonatal and adult chondrocytes along with bone marrow stromal cells (HBMSCs) to differentiate the factors that promote chondrogenesis and maintain functional homeostasis.

Material and Methods: Adult chondrocytes, neonatal chondrocytes and HBMSCs were cultured in monolayers for 1, 2 and 3 weeks in basal or chondrogenic media. Expression of transcription factor Sox9, Aggrecan (ACAN) and Collagen type II (COL2A)was compared via real time polymerase chain reaction (q-PCR). Alternatively, cells were seeded onto 3D PLGA scaffolds and cultured in vitro for 3 and 6 weeks in basal or chondrogenic media. Paraffin sections of the constructs were stained with Alcian blue/ Sirius red and expression of Collagen type II and Aggrecan was visualised via immunohistochemistry.

Results: For monolayer cultures of all three cell types, at week 1, expression of all three genes was down regulated in basal medium compared to levels in chondrogenic medium. By week 2, q-PCR revealed an increased expression of Col2A in chondroinduced neonatal chondrocytes compared to adult chondrocytes and HBMSCs. A steady increase in SOX9 expression was observed with time in all three cell types in chondrogenic medium. However, SOX9 expression in week 2 was higher for each cell type in basal medium compared with chondrogenic medium. ACAN expression by HBMSCs was greatly enhanced compared with that of neonatal and adult chondrocytes after 2 weeks in chondrogenic medium. By week 3, basal cultures of all cell types showed an overall lower level of gene expression compared with chondroinduced cells. 3D constructs revealed the formation of cartilage like tissue for all three cell types with the presence of a prominent superficial layer and middle zone in the chondroinduced constructs. A superficial layer was also observed in constructs cultured in basal media but there was no evidence of any other characteristic zones. A fibrous capsule had formed around the chondroinduced tissue by week 6. Thinnest capsules were observed for constructs seeded with neonatal cells, with thickest capsules in constructs seeded with HBMSCs. Immunohistochemistry revealed a greater presence of aggrecan and type II collagen in the chondroinduced constructs compared to those cultures in basal media.

Conclusion: This comparative study indicates a major difference between the microenvironment of human neonatal chondrocytes, adult chondrocytes and HBMSCs. The expression of high amounts of COL2A and ACAN (considered to be middle to late markers in chondrogenesis) in week 1 in neonatal chondrocytes indicates a difference in temporal gene expression during chondrogenesis or in maintaining cartilage homeostasis. The study provides potentially useful information to inform cell-based therapies for cartilage regeneration.

Articular cartilage has limited regenerative potential. Regeneration via autografts or cell therapy is clinically efficacious but the extent of regenerative success depends upon use of an appropriate cell source. The aim of this study was to compare the proliferative and chondrogenic potentials of three human cell types (human bone marrow stromal cells - HBMSCs, neonatal and adult chondrocytes) commonly used in cartilage tissue engineering.

HBMSCs, neonatal and adult chondrocytes (passage 2) were cultured in basal and chondrogenic media. At 2, 4 and 6 days, the cells were analysed for morphology and doubling time. Alkaline phosphatase specific activity (ALPSA) was quantified for each group at 2, 4 and 6 weeks. Chondrogenic potential of each cell type was assessed via a pellet culture model. Cryosections were stained with Alcian blue/Sirius Red.

HBMSCs showed either elongated or polymorphic phenotypes, with a doubling time of 40 h. Neonatal chondrocytes showed a uniform spindle shape and had the shortest doubling time (16 h). Adult chondrocytes, were also spindle shaped, though slightly larger than the neonatal cells, with a longer doubling time of 22 h. Expression of ALPSA in basal media was of the order HBMSCs > adult chondrocytes > , neonatal chondrocytes. In chondrogenic culture, this order changed to adult chondrocytes > HBMSCs > neonatal chondrocytes. In 3D pellet cultures, all three cell types stained positive for Alcian Blue and showed the presence of chondrocyte-like cells enclosed in lacunae.

This comparative study suggests that neonatal chondrocytes are the most proliferative with lowest ALP expression. However, in terms of clinical applications, HBMSCs may be better for cartilage regeneration given their lower ALP expression under chondrogenic conditions when compared with adult chondrocytes under the same conditions. The study has provided information to inform clinical cell therapy for cartilage regeneration.