Laser-Enhanced Biomorphic Scaffolds Support Multipotent Stem Cell Differentiation and Angiogenesis for Vascularised Bone Regeneration

Abstract

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.

1. Introduction

A novel rattan wood-derived scaffold (GreenBone) composed of non-sintered hydroxyapatite and beta-tricalcium phosphate (β-TCP) demonstrated the ability to support cellular attachment, proliferation, and mineralisation—key processes essential for effective bone repair. Unlike conventional synthetic bone grafts that rely heavily on mined minerals, GreenBone scaffolds are fabricated through a biomorphic transformation process, converting naturally occurring rattan wood into a mineralised structure, substantially reducing the environmental impact associated with raw material extraction. Conformité Européenne (CE) mark approval within the European Union confirms that GreenBone scaffolds meet stringent regulatory standards for safety, biocompatibility, and environmental compliance, supporting their clinical applicability and relevance in translational regenerative medicine [1,2]. The effectiveness of the scaffolds has been validated in various clinical applications, such as reconstructive extremities, demonstrating excellent radiographic integration, biomechanical stability, and minimal post-operative complications when filling bone defects [3]. Similarly, the scaffold provided strong structural support in tibial plateau fractures, promoting bone healing without adverse events, and exhibited encouraging intraoperative handling [4]. The ability to precisely customise the scaffold geometry to match defect dimensions enhances practical applications and has led to optimal surgical outcomes [5]. These attributes highlight GreenBone scaffolds as a promising and versatile tool in addressing critical-sized defects and load-bearing orthopaedic challenges [6].

The biomorphic transformation process for fabricating GreenBone scaffolds aimed towards retaining the hierarchical microarchitecture of natural bone, which provided a strong foundation for tissue regeneration [7]. The scaffolds feature elongated axial channels with lumen diameters ranging from 10 to 500 µm and an average diameter of approximately 300 µm. The asymmetric axial pores, which constitute approximately 60% of the scaffold’s total volume, closely mimic the natural osteogenic architecture, promoting a biomimetic environment conducive to bone regeneration [5]. However, poor interconnection between the scaffold’s axial channels can restrict cell movement and growth, limiting even cell distribution and the formation of new blood vessels, which are important for regenerating large-scale tissue [8,9]. Enhancing channel interconnectivity could significantly optimise the scaffold’s biological performance by facilitating improved nutrient diffusion, cellular infiltration, and angiogenesis, improving the quality of newly formed tissue [10]. The modifications are likely to be particularly beneficial for addressing critical-sized defects and load-bearing applications, where a combination of biological and mechanical performance is essential [11,12].

Porous scaffolds are integral to bone tissue engineering, providing a three-dimensional framework that emulates the extracellular matrix (ECM) of natural bone. The interconnected porous architecture of potential bone scaffolds should facilitate efficient nutrient and oxygen diffusion, which is vital for cell survival and function. Recent studies have confirmed that scaffolds with macropores ranging from 200 to 500 µm are particularly effective in promoting new bone tissue ingrowth and facilitating the transport of body fluids [13]. Scaffold porosity is a key factor in supporting vascular ingrowth, which underpins bone integration and long-term tissue maintenance. Sufficient pore size and connectivity enable blood vessels to infiltrate the scaffold, delivering nutrients and clearing metabolic waste during regeneration. Vascularisation is vital for delivering nutrients and removing waste products, supporting the metabolic needs of proliferating and differentiating cells within the scaffold [1]. Therefore, the design of scaffolds with appropriate macro- and microporosity is essential for regenerative medicine, as scaffolds provide the necessary mechanical support and create a conducive environment for cellular activities and vascularisation, ultimately leading to successful bone regeneration.

Our previous study investigated the gene expression profiles and angiogenic potential of stem cells on unmodified GreenBone scaffolds in vitro [14]. Following on these promising results, a subsequent study aimed to explore the potential benefits of enhanced pore interconnectivity on improving stem cell proliferation on GreenBone scaffolds. Water jet laser-drilling was employed for drilling channels perpendicular to the axial porosity of the scaffolds, enhancing the volumetric porosity and pore interconnectivity in the GreenBone scaffolds by 22.5 vol.%, and the laser-drilled scaffolds were examined for toxicity using the G292 cell line [5]. In this study, we provide a potentially clinically relevant multi-cellular biological assessment of our porosity engineered GreenBone CE marked medical devices using patient-derived BMMSCs, culture-expanded MSCs, and endothelial cells. We investigated whether the enhanced pore interconnectivity and architecture of the scaffolds could drive a depth-dependent cellular infiltration inducing a non-linear temporal gene expression response from key osteogenic (BMP2, RUNX2, COL1A1), matrix-remodelling (MMP9, TIMP1), and angiogenesis (VEGFA) genes. The integration of sustainable biomaterials processed using laser-modification techniques offers a promising avenue for improving scaffold performance in complex clinical scenarios, addressing a critical gap in translating biomorphic ceramic scaffolds toward vascularised, clinically relevant bone repair, particularly in cases where load-bearing capacity is necessary for the biomechanical stabilisation of the fracture site.

2. Materials and Methods

2.1. Scaffolds Processing

The flow chart of the experimental procedure is displayed in Figure 1. GreenBone scaffolds (Ø = 10 mm, H = 4 mm) were obtained from GreenBone Ortho (Faenza, Italy) with a reported chemical composition of 85% hydroxyapatite (Ca₅(PO₄)₃OH) and 15% β-TCP (Ca₃(PO₄)₂) [1,2]. The biomorphic apatite scaffold was also doped with Mg²⁺ and Sr²⁺.

A SYNOVA MCS300 Nd:YAG pulsed water jet laser, operating at a wavelength of 532 nm, a pulse frequency of 6 kHz, and a pulse width of 250 ns (FWHM), was run at 31 W and 300 bar to drill channels of approximately 700 µm in diameter, perpendicular to the longitudinal porosity of the GreenBone scaffolds. The laser-drilling process enhanced pore interconnectivity, thereby improving microfluidic flow for cell proliferation while minimising thermal damage to the scaffolds [5].

2.2. Scaffold Characterisation

A Hitachi SU8230 scanning electron microscope (SEM) and energy-dispersive X-ray spectroscopy (EDS) operating at 10 kV and 20 kV in both backscattered and secondary electron imaging modes were employed for scaffold surface characterisation. Carbon paint was applied to each scaffold prior to imaging to minimise charging.

The GreenBone scaffolds were structurally analysed using a Skyscan 1172 (Bruker, Kontich, Belgium) micro-computed tomography (µCT) system. The X-ray source operated at a constant voltage of 100 kV and a current of 100 µA, with two 0.5 mm aluminium filters applied to optimise image quality. Scanning was performed with a rotational step of 0.4°, resulting in a total scanning time of 3 h per sample. The three-dimensional (3D) structure of the scaffolds was reconstructed from the recorded 2D projection images using NRecon V2 software and further analysed with SkyScan software (Skyscan1172 version 1.5.26.0).

2.3. In Vitro Studies

The GreenBone scaffolds (Ø = 10 mm, H = 4 mm) were first sterilised by X-ray sterilisation at 25 kGy, followed by surface sterilisation with 70% (v/v) ethanol, rinsing in Dulbecco’s Phosphate-buffered saline (DPBS), and exposure to UV light for 20 min. Scaffolds were ‘primed’ in StemMACS (SM) medium under standard culture conditions (37 °C, 5% CO₂) for 72 h to help provide the scaffolds with the cellular attachment factors present in the SM medium [15]. A schematic of the experimental workflow for the in vitro studies is shown in Figure 2.

2.3.1. Ethics and Harvesting of Bone Marrow Aspirate

Bone marrow aspirates (BMAs) were harvested from n = 10 patients (mean age: 45 years, range 28–79 years) undergoing elective orthopaedic surgery for fracture non-union at the Trauma & Orthopaedic Unit at Leeds General Infirmary (LGI), Leeds, UK,

Table 1. Donor characteristics and allocation to experimental workflow.

Sex	Age	Cultured BM MSC (cMSC)		BMA
		SEM and Confocal Imaging	cMSC and HUVEC Co-Culture	CFU-F Assay and qPCR
		n = 3	n = 4	n = 7
Male	29	Yes	Yes
Male	37	Yes	Yes
Male	29	Yes	Yes
Female	33		Yes	Yes
Female	79			Yes
Female	44			Yes
Male	67			Yes
Male	65			Yes
Female	42			Yes
Male	28			Yes
Mean Age	45	32	32	51

. Approximately 8 mL of BMA was collected per donor into tubes containing ethylenediamine tetraacetic acid (EDTA) to inhibit coagulation. Ethical approval was granted by the National Research Ethics Committee (REC) Yorkshire and The Humber—Leeds East to procure human donor samples (REC approval number 18/YH/0166). Informed written consent was given by all patients included in this study.

2.3.2. Cultured Mesenchymal Stromal Cells (cMSC) Seeded on GreenBone

Following RBC lysis of BMA (n = 4), nucleated cells were cultured in StemMACS MSC Expansion Media (SM, Miltenyi Biotec, Surrey, UK) to obtain passage 3 (P3) culture-expanded BM-MSCs (cMSC) using established adherence culture methods [16]. Preconditioned GreenBone scaffolds were seeded with P3 cMSCs (4 × 10⁴ cells per scaffold, n = 3 donors, mean age: 32 years, Table 1. After an initial incubation of 3 h, the scaffolds were transferred to low-attachment 24-well plates and cultured for 7 days with half-SM media washes and then processed for imaging.

SEM Imaging

The microstructure of the cell-seeded GreenBone scaffolds was analysed using SEM (Hitachi SU8230 scanning electron microscope, Hitachi UK, Maidenhead, UK) operating at 10 and 20 kV in backscattered and secondary electrons imaging modes. The cells were fixed using formaldehyde and dehydrated using serial alcohol concentrations (50% to 100%) for 15 min each. The scaffolds were mounted on a conductive carbon sticker, and conductive carbon paint was utilised to prevent the scaffolds from charging.

Confocal Imaging

Non-adherent cells were removed with two DPBS washes, and adherent cells were fixed with 4% (v/v) neutral-buffered formalin for 15 min. The samples were then permeabilised with 0.1% (v/v) Triton X-100 (Sigma-Aldrich, Germany) for 5 min and rinsed twice with DPBS. Actin filaments were stained with Alexa Fluor™ 488-conjugated phalloidin (Invitrogen, CAT no: A12379, Waltham, MA, USA) for 2 h at room temperature in the dark. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) at 1 µg/mL for 30 min. Samples were washed with DPBS to remove excess dye and reduce background fluorescence. Imaging was performed using a Leica TCS SP8 confocal microscope with appropriate excitation and emission filters for Alexa Fluor™ 488 (488 nm) and DAPI (405 nm).

2.3.3. Co-Culture of cMSC with HUVECs

Human umbilical vein endothelial cells (HUVECs; Promocell, Heidelberg, Germany) were cultured to confluence in T75 flasks with endothelial growth media (Promocell) and seeded onto primed GreenBone scaffolds at a 1:1 ratio with P3 cMSCs (n = 4 donors, mean age: 32 years, Table 1); a total of 2 × 10⁵ cells were seeded. A 1:1 mixture of endothelial and SM media was used to support both cell types. Control scaffolds were seeded exclusively with 2 × 10⁵ BMMSCs per scaffold.

Supernatants were collected on days 1, 7, and 14 and stored at −80 °C until analysis. Media was removed and replaced with fresh media 24 h prior to supernatant collection to allow for 24 h of cytokine release at each time point. Supernatants were collected on days 1, 7, and 14 and stored at −80 °C until analysis. Media changes were performed on day 1 (following supernatant collection), followed by semi-weekly half-media replacements. VEGF levels were measured using an ELISA according to the manufacturer’s instructions. The absorbance was measured at 450 nm using a Cytation 5 imaging plate reader (BioTek, Thorold, ON, Canada) manufactured by Agilent Technologies (United States of America).

2.3.4. Bone Marrow Aspirate Seeded on GreenBone Scaffolds

BMA was harvested as previously described (n = 7 donors, mean age: 51 years, Table 1) and filtered through a 70 µm cell strainer (Falcon, Fisher Scientific, Loughborough, UK) to exclude fat or bone debris. Primed scaffolds were seeded with 316 µL of filtered BMA and incubated for 3 h under standard culture conditions. Post BMA incubation, scaffolds were transferred to 24-well low-attachment plates (Corning, NY, USA), where 1.5 mL of SM media was added to each well and incubated for 48 h. The media was then carefully replaced with 1.5 mL of fresh SM media. The scaffolds were cultured for 1, 2, and 4 weeks, with bi-weekly half-media changes [9,15]. The cells isolated from BMA will henceforth be referred to as BMMSCs. The BMMSCs were characterised for trilineage differentiation (Supplementary Figure S1).

Colony Forming Unit-Fibroblast (CFU-F)

The CFU-F assay evaluated BMMSC attachment to GreenBone scaffolds derived from fresh BMA. In brief, 200 μL of BMA was seeded in duplicate into 100 mm Petri dishes (Corning, New York, NY, USA) containing 10 mL of SM medium and incubated at 37 °C with 5% CO₂. After 48 h, non-adherent cells were removed and cultured in 100 mm Petri dishes. The adherent cells were cultured for 14 days, with half-medium changes performed weekly. The resulting colonies were fixed with 3.7% formaldehyde for 10 min and stained with 1% (w/v) methylene blue for 30 min. The excess stain was removed with deionised water, and the dishes were air-dried before colony counting. The number of attached BMMSCs to scaffolds was determined by subtracting the CFU-F counts of the scaffold-seeded dishes from the control dishes without scaffolds, respectively.

Procedure for Cell Harvesting from Scaffolds

At each time point, cells from one of the scaffold duplicates were harvested to test for BMMSC survival following the trypsin/collagenase protocol [9]. The media was aspirated and the scaffolds were washed thrice with DPBS, followed by 1% (v/v) trypsin solution (Life Technologies, Paisley, UK) to cover the scaffold and incubated for 5–10 min. The trypsinised solution, containing the detached cells, was filtered into a separate 15 mL Falcon tube to exclude any scaffold debris, and the trypsin activity was neutralised by 5 mL of DMEM media. The solution was centrifuged, the supernatant was removed, and the cell pellet was resuspended in several mL of media. Tightly adherent cells were detached by further treating the scaffolds with 0.6 mL of collagenase (Stem Cell Technologies, Vancouver, BC, Canada). The scaffolds were incubated at 37 °C for 30 min, with manual agitation every 10 min to enhance enzymatic activity. Following incubation, the solution was filtered through a 70 µm cell strainer and combined with the previously collected cell pellet suspended in media. The suspension was centrifuged to remove residual collagenase, and the resulting cell pellet was resuspended in 10 mL of fresh media. The released cells were counted using the trypan blue exclusion assay and subsequently cryopreserved.

Gene Expression

At each time point, the cells were lysed using Buffer RL (350 µL) and vortexed over ice. RNA was extracted using the Total RNA Purification Kit (Norgen Biotek, Thorold, ON, Canada), following the manufacturer’s instructions. The purity and concentration of the RNA were evaluated using a NanoDrop™ One Microvolume UV–Vis Spectrophotometer (Thermo Scientific). RNA aliquots were reverse-transcribed into complementary DNA (cDNA) using RT master mix (Fluidigm, CA, USA). The reaction was performed in a Thermal Cycler 480 (Applied Biosystems, CA, USA) under the following conditions: 5 min at 25 °C, 30 min at 42 °C, 5 min at 85 °C, followed by a hold at 4 °C. The cDNA underwent 19 cycles of pre-amplification with Taqman assays using a PA master mix Fluidigm (Fluidigm, San Francisco, CA, USA), following the manufacturer’s guidelines. The complete list of TaqMan probes is provided in Supplementary Table S1. Quantitative polymerase chain reaction (qPCR) was performed using the Fluidigm 48.48 Dynamic Array™ integrated fluidic circuit (IFC, Fluidigm) on the BioMark HD system, as detailed in [17]. Gene expression levels were normalised to Hypoxanthine phosphoribosyl transferase1 (HPRT1) and calculated for relative expression using the 2^−ΔCt method, where ΔCt = Ct target gene—Ct housekeeping gene.

2.3.5. Statistics

All statistical analyses were conducted using GraphPad Prism software (version 9.5.1). Data normality was assessed using the Shapiro–Wilk and Kolmogorov–Smirnov tests. For data following a normal distribution, comparisons of means were performed using either the unpaired t-test or ordinary analysis of variance (ANOVA). Results were presented as mean ± standard deviation, and statistical significance was defined as p ≤ 0.05. The following symbols were used to denote the levels of significance: “ns” denotes no significance, “*” for p < 0.05, “**” for p < 0.01, “***” for p < 0.001, and “****” for p < 0.0001.

3. Results

3.1. Scaffold Characterisation Post Laser-Drilling Processing

The laser-drilled cross-sectional holes (Figure 3A) exhibited precise and well-defined cylindrical geometries without disruption to the natural longitudinal porosity of GreenBone scaffolds (Figure 3B,C) formed during biomorphic fabrication, signifying that the scaffolds remain structurally stable despite the external drilling procedure. EDS elemental mapping highlights a homogenous distribution of key elements—calcium (Ca), Oxygen (O), Phosphorus (P), Strontium (Sr), and Magnesium (Mg)—across both drilled and undrilled regions (Figure 3A−C), demonstrating that the drilling process preserved the scaffold’s chemical composition without elemental depletion or redistribution. The porous, highly irregular, and textured scaffold’s appearance (Figure 3D) enhances the surface area, presenting positive implications for the scaffold’s ability to promote cell adhesion. The false-colour topography map (Figure 3E) emphasises the scaffold’s exterior texture and interconnected surface pores, with height variations ranging between ~25 µm and 100 µm across the scaffold. The laser-drilled scaffolds demonstrated a surface roughness of 12.4 µm, suitable for promoting osteogenic differentiation of bone marrow stem cells (10 to 15 µm diameter) on the surface of the GreenBone scaffolds [18].

Micro-computed tomography (µCT) analysis of undrilled and laser-drilled GreenBone scaffolds (Figure 3F) depicts the elongated, cylindrical axially aligned channels as a result of the scaffold fabrication process. The average diameters were ~300 µm [5], visible in the coronal view as longitudinal channels and the transverse view as circular cross-sections. Limited interconnectivity between the natural lateral channels, as shown in the undrilled scaffolds, has been found to confine cell migration and proliferation [9]. The µCT reconstruction confirms that the laser-drilling process created uniform, aligned, perpendicular channels bridging the previously unconnected natural channels, enhancing the scaffold’s interconnectivity without evidence of material collapse or deformation (Figure 3G).

3.2. Culture of cMSCs on Drilled GreenBone Scaffolds

Figure 4A show the structural characteristics of GreenBone scaffolds from the side and top views, respectively, before BMMSC seeding. The lower-magnification SEM image (Figure 4A) reveals the porous structure with well-defined top surface macropores. The scaffold’s surface exhibits irregular morphology with rough textures where several thin, fibrous cell structures extend across the voids, confirming the presence of cellular projections and matrix deposition after 7 days in culture (Figure 4B). Several elongated cytoplasmic extensions bridge the scaffold walls and extend between the pore’s edges, forming a network structure. The small, fibrous deposits are seen along the scaffold’s walls, indicating the presence of ECM components secreted by the cells. In addition, the observed cellular extensions suggest that BMMSCs have proliferated within the scaffold, infiltrating deeper regions.

Figure 4C illustrate the BMMSCs cultured on GreenBone scaffolds for 7 days, stained with Phalloidin (green) and DAPI (blue) to assess actin filament to show cell morphology and attachment onto the laser-drilled GreenBone scaffolds. The fluorescence imaging reveals well-defined actin networks, indicative of cytoskeletal organisation essential for cell attachment and spreading. The nuclei appear uniformly distributed, suggesting a healthy and proliferative cell population. Cells are observed to be evenly dispersed across the scaffold’s surface, with no visible areas of detachment or aggregation. The attached cells’ consistent morphology and actin filaments indicate that the scaffold promotes strong adhesion and cytoskeletal integrity. The 3D reconstructed view (Figure 4C, bottom image) reveals evidence of cellular migration into the scaffold, with cellular infiltration extending up to 300 µm. This could be either from initial attachment from day 0 or from migration over 7 days in culture into the longitudinal pores of the scaffold.

3.3. Co-Culture of HUVECs and cMSCs on Drilled GreenBone Scaffolds

At day 1, VEGF production is low across all conditions, with no significant differences observed between the scaffold-only and co-culture groups (Figure 4D). By day 7, VEGF levels rise significantly in the co-culture condition compared to BMMSCs alone (p < 0.0001) and also show a significant increase within the co-culture group between day 1 and day 7 (p < 0.01). At day 14, VEGF levels in the co-culture group decline significantly lower than in the BMMSC-only condition (p < 0.01). In contrast, VEGF levels in BMMSCs alone increase significantly between day 7 and day 14 (p < 0.001), while levels in the co-culture group decrease (p < 0.01). Overall, BMMSCs alone exhibit a significant increase in VEGF secretion over the 14-day period.

3.4. BMA Attachment and Survival on Drilled GreenBone Scaffolds

The colony-forming unit (CFU-F) assay (Figure 5A,B) reveals a progressive increase in BMMSC attachment and proliferation on GreenBone scaffolds, indicating a time-dependent growth trend over the four-week study period. A colony is defined as having at least 50 cells. At 72 h, CFU-F counts were the lowest, with colony numbers ranging from 0 to 9 across samples, suggesting limited initial cell adhesion [19]. By week 1, a modest increase in CFU-F counts was observed (from 0 to 24 CFU-F per sample), although substantial inter-sample variability was evident, as reflected by the standard deviation.

At week 2, the median CFU-F count continued to rise (from 1 to 41 CFU-F per sample), indicating a trend of improved adhesion, possible early proliferation, and early colony expansion. By week 4, CFU-F attachment significantly increased, reaching its highest levels (from 1 to 70 CFU-F per sample). Statistical analysis indicated no significant differences among 72 h, week 1, and week 2, whereas week 4 exhibited a statistically significant increase in CFU-F formation compared to earlier time points (p < 0.01). The high variability of patient-derived BMMSCs was expected due to differences in donor variations and scaffold microenvironments.

Gene Expression

The relative gene expression (to the housekeeping gene, HPRT1) of BMMSCs cultured on laser-drilled GreenBone scaffolds was analysed at three time points to assess the dynamic temporal changes in gene expression (Figure 5C). At week 1, BMMSCs expressed 44 out of 48 genes (91%), with several genes showing moderate to high relative expression. Notable genes include COL1A1, TGFBR2, and VEGFA, which indicate the active cellular processes during the initial phase of culture. At week 2, overall gene expression decreased significantly, with only 25 out of 48 genes (52%) showing detectable levels; this reduction was consistent across most transcripts, with lower relative expressions observed for key genes such as TIMP1, MMP9, and VEGFA compared to week 1. The intermediate phase marked a noticeable decline in overall cellular activity. By week 4, gene expression levels rebounded, with 47 out of 48 genes (97%) being expressed. Many genes showed peak expression, including TIMP1, TIMP2, MMP9, P21, and PTPRC. P16 was seen to be statistically significant when comparing week 2 and 4, but significantly reduced with time (p = 0.028). Of note, three other transcripts approached significance for comparisons of week 1 and 2, TIMP1 (p = 0.058), FGFR1 (p = 0.0530) and FGFR2 (p = 0.0817); additionally, comparing week 2 and 4 was FGFR1 (p = 0.0891). VEGFA maintained consistent expression across all time points, although relative expression levels increased by week 4. The rebound reflected increased activity in a broad range of transcripts. The data shows an initial high level of gene expression at week 1, a significant decrease at week 2, and a resurgence at week 4, with distinct genes peaking at different stages.

4. Discussion

4.1. Engineering Characterisation of the Scaffolds

In this study, we investigated cell proliferation and the osteogenic and angiogenic differentiation of bone marrow stem cells on laser-drilled GreenBone scaffolds. Water jet laser-drilling was employed to enhance the volumetric and interconnected porosity of the biomorphic hydroxyapatite scaffolds [5]. The unmodified GreenBone scaffolds, consisting of 300 µm axial pores and presenting 57 vol.% porosity, demonstrated 22.5 vol.% volumetric porosity increase after water jet laser-drilling [5]. The inherent anisotropic structure of the GreenBone scaffolds resembles the structure of the osteonic system [5,7]. The 4-through laser-drilled holes (700 µm) were drilled perpendicular to the natural porosity of the biomimetic scaffolds, improving pore interconnectivity without compromising the structural integrity of the material [5], also confirmed by SEM and µCT analysis in this research. The laser-drilled scaffolds presented improved cellular proliferation, migration, gene expression profiles, and angiogenic potential compared to the unmodified GreenBone scaffolds [9]. The surface roughness of the laser-drilled scaffolds was measured as 12.4 µm, which aligns with the findings by Daskalakis et al. (14.5 µm) [5]. Laser-drilling did not obstruct or block the natural porosity of the scaffolds, as the water jet removed the debris generated during processing and prevented thermal build up in the drilled zone [5]. EDS mapping confirmed that the lack of laser-drilling induced contamination in the scaffolds due to the consistency of the element’s distribution across the non-drilled and laser-drilled parts of the material. EDS analysis identified calcium (Ca), Oxygen (O), Phosphorus (P), Strontium (Sr), and Magnesium (Mg), which correspond to the previously reported composition of the GreenBone scaffolds (85% hydroxyapatite (Ca₅(PO₄)₃OH) and 15% β-TCP (Ca₃(PO₄)₂)) [5,7,9]. The scaffolds were also doped with Mg²⁺ and Sr²⁺ ions for enhancing the biocompatibility, promoting osteogenesis, and improving the mechanical strength of the scaffolds [5,7,9]. GreenBone scaffolds presented a crystalline sub-micron hydroxyapatite structure [5,7] due to the low-temperature employed during the manufacturing process, in contrast to conventional bio-ceramics manufactured through sintering (Engipore™; Fin-Ceramica Faenza, Italy) (HA ∼5 wt% of β-TCP, macropore size 100–200 μm, and porosity ∼60 vol.%.) [5,7]. Additionally, the compressive strength of the unmodified GreenBone scaffolds in (i) Sprio et al. [1] (9.8 ± 5.9 MPa) and (ii) Daskalakis et al. (10.21 ± 0.55) [5], along with the compressive strength of the laser-drilled GreenBone scaffolds in this research (7.39 ± 0.18), were superior to commercial sintered scaffolds by Kasios^®, Atoll (HA-β-TCP ∼25%, porosity 64 ± 4 vol.%), 5.7 ± 1.6 MPa [5].

4.2. Gene Expression

Evaluation of gene expression with BMMSCs provides complementary insights into cellular behaviour. BMA reflects the physiological microenvironment in the bone marrow and was thus used to evaluate baseline gene expression. BMA contained a heterogeneous cell population and offered an in vivo-like context, reflecting the natural microenvironment and baseline gene expression under physiological or pathological conditions. This approach provided insights into clinically relevant, physiologically diverse cellular responses [20].

Conversely, cMSCs offered a more homogeneous, controlled system for detailed analyses of cells behaviour, differentiation potential, and scaffold interactions [21]. Pore size and pattern enhances the nutrient infiltration providing biomimetic and supportive environment for cell culture, their survival, proliferation, and angiogenic and osteogenic differentiation [22]. Laser-drilled GreenBone scaffolds with enhanced volumetric and interconnecting porosity provided a microenvironment for improved cell growth. Integrating both cell types strengthened the validation of in vitro findings, aligning experimental outcomes more closely with in vivo complexity and clinical applications.

The elevated expression levels of BMP2 and VEGFC in cMSCs compared to BMMSCs within laser-drilled scaffolds suggest the enhanced osteogenic and angiogenic potential of cMSCs, indicating their suitability for bone regeneration applications. The improved interconnectivity of the pores in laser-drilled GreenBone scaffolds likely facilitated nutrient diffusion, waste removal, and cell–cell interactions, which promoted the activation of genes critical for osteogenesis and vascular support [14]. BMMSCs, on the other hand, exhibited gene expression patterns in the drilled scaffolds, characterised by the upregulation of genes involved in tissue remodelling and immunomodulation, indicating that BMMSCs may add to the repair process through Extracellular Matrix (ECM) regulation and modulation of inflammatory responses. The osteogenic (BMP2, RUNX2, COL1A1) and ECM-remodelling (MMP9, TIMP1) genes followed a non-linear trajectory, where early upregulation took place in the first week, which was followed by a transient suppression at week 2 and a rebound by week 4. The rebound trend observed suggests a matrix-mediated cellular adaptation rather than continuous or monotonic differentiation, which is consistent with early bone healing phases where remodelling precedes maturation [14]. This temporal pattern implies the presence of early in vivo bone healing, where an initial proliferative and inflammatory phase is followed by a brief remodelling or quiescent period before the renewed osteogenic activity and matrix maturation during the reparative phase takes place. Clear cell-source-specific responses were observed, with cMSCs exhibiting stronger osteogenic commitment, while BMMSCs showed enhanced ECM remodelling and immunomodulatory gene expression. This emphasised the complementary regenerative roles of BMMSCs driven by scaffold architecture. These differences are consistent with the inherent biological functions of the two cell types, as cMSCs appeared to be more specialised for bone formation and vascularisation, whereas BMMSCs contributed more broadly to tissue repair and immune-related processes [23]. The variability in gene expression between cMSCs and BMMSCs in the drilled scaffolds may resulted from differences in cellular phenotype, scaffold architecture, or culture conditions. The laser-drilled design of the scaffolds enhanced microenvironmental conditions, such as interconnectivity and fluid flow, which modulated gene expression [24]. The findings emphasise the potential of cMSCs for targeted bone regeneration therapies, such as bone grafting or osteogenic repair, while highlighting the broader applicability of BMMSCs in tissue engineering applications requiring structural and immune support [25].

In contrast, the GreenBone scaffolds without drilled channels, studied by Ganguly et al. [17] showed a slower, more gradual cellular response, with distinct gene expression patterns. In these scaffolds, BMMSCs expressed high levels of ECM-associated proteins (SPARC, SPP1) and mesenchymal markers (NT5E, THY1). However, it showed a tilt toward adipogenesis, indicated by elevated FABP4 expression and the absence of Sox9, a key transcription factor for chondrogenesis [26]. Furthermore, scaffolds without holes retained haematopoietic-lineage cells, marked by PTPRC expression, which may contribute to bone regeneration through paracrine signalling [27]. While both scaffold types supported ECM remodelling and tissue regeneration, scaffolds with holes were more effective in promoting rapid osteogenesis and vascularisation, whereas scaffolds without holes provided a more gradual and controlled regenerative environment suitable for steady tissue remodelling [28].

We observed cellular ingression in the laser-drilled biomorphic GreenBone scaffold. Further investigation of the materials is needed to compare laser-drilled to the original materials to ascertain whether drilling improves this. As displayed in Figure 5, the cytoskeletal organisation of the cells exhibited well-organised and densely distributed actin filaments surrounding the interconnected pores of the scaffold. The arrangement reflects strong cell–matrix interactions, which are critical for cellular communication and osteogenic differentiation. Such structural organisation has been reported as a key indicator of the scaffold’s ability to provide mechanical and biological cues that promote cell functionality [21,29]. Our findings are consistent with earlier research demonstrating that biomimetic scaffolds with enhanced interconnectivity improve cellular integration and tissue repair outcomes [12].

4.3. Angiogenic Potential

The rise in VEGF levels observed in the co-culture from day 1 to day 7 likely occurred through two main mechanisms. Firstly, endothelial cells produce VEGF, directly promoting angiogenesis and contributing to the higher levels detected in co-culture conditions [30]. Secondly, BMMSCs within the co-culture have the potential to differentiate into endothelial cells under the influence of paracrine signals from endothelial cells; this differentiation enhance the synergistic effect of both cell types, further boosting VEGF production [31]. Previously, VEGF expression was reported to be induced by differentiation of mouse progenitor cells into endothelial cells [32,33]. Paracrine effect been suggested to express localised expression of growth factors, i.e., VEGF through expression of receptors such as VEGFR-2 a process fundamental to vasculogenesis [34]. The rise in VEGF expression was reported to increase from 40 h until 7 days earlier in two dimensional invitro cultures in a time-dependent manner [35,36]. In contrast, VEGF secretion dynamics in BMMSCs alone show a slight reduction from day 1 to day 7, followed by a significant increase by day 14 compared to the co-culture condition. The expression of VEGF was reported earlier to either remains stable or even slightly declines due to lack of stimulatory feedback of neighbouring cells which is the case in co culture cell culture [37]. Moreover, in monolayer single cell culture (BMMSCs), the autocrine signalling could saturate and lead to the downregulation of VEGF expression via a negative feedback loop [38]. As BMMSCs proliferate, increasing cell density can alter VEGF secretion as high cell density and confluence affect cellular behaviour and paracrine signalling, which also influences VEGF levels [39]. Co-culturing endothelial progenitor cells with MSCs stimulates the formation of a mature vascular network over 7–10 days [29,40] and has been shown to enhance endothelial differentiation mediated by VEGF [41]. The reduction expression of VEGF levels observed in the co-culture condition from day 7 to day 14 can likely be due to cellular crowding and contact inhibition, which suppress proliferative and metabolic activity as the co-culture reaches confluence. Additionally, nutrient depletion over time creates a suboptimal microenvironment that impairs VEGF production [42]. VEGF stability also plays a role; despite having a half-life of approximately 8 h, prolonged culture periods without medium replenishment can result in protein degradation and reduced bioavailability [43,44].

A comparison between the water jet laser-drilled GreenBone scaffolds and non-drilled GreenBone scaffolds from previous research [9] revealed distinct VEGF production patterns. Non-drilled scaffolds exhibited a delayed response, with substantial VEGF increases only becoming evident by day 14. The findings suggest that non-drilled scaffolds support a gradual VEGF increase, which could benefit applications requiring sustained and steady vascularisation. In contrast, the scaffolds with drilled holes expressed accelerated and enhanced VEGF production, facilitating improved cell–cell interactions within the co-culture, leading to a significant VEGF increase by day 7. Enhancing the pore interconnectivity of the GreenBone scaffolds not only impacted the magnitude, but also the timing of angiogenic signalling. MSC–HUVEC exhibited a significant early VEGF secretion peak at day 7, whereas angiogenic responses on unmodified scaffolds was more delayed [14]. The accelerated VEGF release was indicative of endothelial stromal crosstalk, essential for vascularisation during bone regeneration. The laser-drilled GreenBone scaffold architecture appeared to synchronise osteogenic and angiogenic processes required for clinically relevant bone repair. However, cell phenotype and qPCR analysis would need to be carried out to further confirm this.

The addition of laser-drilled holes in GreenBone scaffolds enhanced cellular behaviour and the microenvironment, promoting enhanced cellular adhesion. The increased porosity and interconnected channels mimic natural ECM, providing pathways for effective cell migration, infiltration, and population of the scaffold. The structural modifications improved the cell–cell and cell–matrix interactions, both of which are critical for tissue regeneration [45].

Furthermore, the improved microfluidic properties of the drilled scaffolds ensured efficient nutrient and oxygen diffusion, which likely created a favourable environment for cell proliferation and function [46]. The drilled scaffolds supported the accelerated VEGF production by fostering a microenvironment conducive to angiogenic activity. The findings align with previous research demonstrating that scaffold porosity and microarchitecture are essential for modifying cell behaviour and enhancing vascularisation in tissue-engineered constructs [45,47].

4.4. Scaffold Cellular Viability

Fluorescent microscopy revealed uniform cell viability and migration across the GreenBone scaffolds, implying the presence of a microenvironment conducive to cellular adhesion and colonisation. Cellular infiltration was observed to extend up to 300 µm into the scaffold, which demonstrates the effectiveness of the interconnected porous structure in assisting nutrient diffusion and also cellular migration. The cellular migration depth highlights the scaffold’s capacity to support uniform cell distribution, which is crucial for tissue regeneration, particularly in significant bone defects where complete cellular integration throughout the scaffold is essential. The observations align with previous findings highlighting the importance of scaffold porosity and interconnectivity in enhancing cellular migration and nutrient exchange [7,9]. As displayed in Figure 3D, the cytoskeletal organisation of the cells exhibited well-organised and densely distributed actin filaments surrounding the interconnected pores of the scaffold. The arrangement reflects strong cell–matrix interactions, which are critical for cellular communication and osteogenic differentiation. Such structural organisation has been reported as a key indicator of the scaffold’s ability to provide mechanical and biological cues that promote cell functionality [24]. Therefore, supports the scaffold’s suitability for bone tissue engineering applications. In addition, the high cell viability and extensive cellular infiltration observed in the GreenBone scaffolds highlight their potential to encourage osteogenesis and angiogenesis since the interconnected architecture facilitates the migration of osteoblast precursors and creates a microenvironment favourable for vascularisation, both of which are essential for successful bone regeneration. Our findings are consistent with earlier research demonstrating that biomimetic scaffolds with enhanced interconnectivity improve cellular integration and tissue repair outcomes [3,11].

5. Limitations and Future Directions

We are mindful that our study has a limited number of samples and that the time points from week 1 to week 4 are relatively shorter periods, especially considering the average time for bone repair post-defect. However, our future work will focus on increasing the sample size and investigating them for longer periods and time frames to better reflect alignment with the time required for bone healing. Evaluation of the genetic transcripts used in this study aimed at bone formation, regeneration, and differentiation with a larger sample size will provide further insights into the potential mechanisms for bone healing.

Despite these challenges, the findings highlight the potential for laser-drilled GreenBone scaffolds to address critical challenges for bone defect repair, particularly in applications requiring enhanced vascularisation and rapid integration. Future studies will focus on in vivo evaluations to validate the scaffolds’ long-term functional performance and assess their clinical applicability. This work advances the design and application of biomimetic scaffolds, contributing to developing innovative solutions for critical-sized bone defects and complex regenerative medicine challenges.

6. Conclusions

This investigation demonstrates that laser-drilled GreenBone scaffolds exhibited enhanced porosity, cellular infiltration, osteogenic differentiation, and angiogenic potential, with the architecture of the scaffolds actively modulating biological responses beyond structural modification alone.

SEM, µCT, and EDS verified that the water jet laser-drilling introduced well-defined lateral micro-channels, significantly increasing porosity and interconnectivity. The mechanical integrity and elemental composition were not compromised. The modifications facilitated improved nutrient diffusion and cellular migration, essential for tissue regeneration.

BMMSCs and cMSCs maintained >90% viability on laser-modified scaffolds and increased colony formation and proliferation over four weeks. Confocal microscopy verified deep cell penetration (~300 µm) and extensive cytoskeletal organisation, representing biologically relevant early integration depth under static in vitro conditions and being consistent with the values reported for other porous ceramic scaffolds.

BMMSCs retained their osteogenic, chondrogenic, and adipogenic differentiation capacity, confirmed using histological staining (Alizarin Red, Alcian Blue, and Oil Red O). The results confirm the scaffold’s ability to support multipotent cell differentiation, a critical factor in bone regeneration.

Quantitative PCR revealed the upregulation of osteogenic markers (BMP2, RUNX2, COL1A1) and angiogenic factors (VEGFC, TIMP1, MMP9), particularly at week 4. The non-linear temporal gene expression pattern, characterised by an early response, a transient dip, and a late rebound, suggests matrix-driven cellular adaptation consistent with early in vivo bone healing dynamics.

VEGF secretion increased, particularly in MSC-endothelial cell co-cultures at day 7, highlighting the scaffold’s capacity to promote early angiogenic signalling and endothelial cell recruitment, which are essential for vascularised bone regeneration.