3D Environment with BMP-2-Releasing Nanocarriers Enhances Osteogenic Commitment of Human Tendon Stem Cells

Technical Review | Biomedical Materials, 2026 | Lamparelli, Lancellotti, Manzo, Cortella et al. | Featured Tool: PreciGenome iFlow™ Microfluidic Platform

Background

Bone-tendon interface engineering remains a significant clinical challenge, driven by the need for innovative strategies that faithfully replicate the complex transitional tissue between tendon and bone. Current approaches often rely on bone marrow-derived mesenchymal stem cells (MSCs) or immortalized cell lines, which may not reflect the specific mechanobiology and differentiation potential of cells native to the tendon-bone junction.

Human tendon stem/progenitor cells (hTSPCs) represent a clinically relevant, tissue-specific primary cell population with demonstrated multipotency — capable of osteogenic, chondrogenic, and adipogenic differentiation. However, the osteogenic differentiation of primary hTSPCs within a bioprinted, growth factor-releasing three-dimensional microenvironment had not been previously explored.

Bone Morphogenetic Protein-2 (BMP-2) is a potent osteoinductive growth factor, but its therapeutic utility is limited by rapid degradation and short half-life in vivo. PLGA nanocarriers offer a solution by protecting BMP-2 from premature degradation while enabling controlled, sustained release. This study combined BMP-2-loaded PLGA nanocarriers with 3D bioprinted GelMA scaffolds under dynamic perfusion to create an osteoinductive microenvironment for hTSPCs — a first-of-its-kind platform for tendon-bone interface tissue engineering.

Figure 1. Physicochemical characterization of PLGA nanocarriers (PLGA-NCs). (a) Dynamic light scattering analysis showing hydrodynamic mean diameter and polydispersity index of BMP-2–loaded and unloaded formulations. (b) Corresponding ζ-potential measurements. (c) SEM images confirming spherical morphology with narrow size distribution. (d) Cumulative BMP-2 release profiles from PLGA-NCs in sink conditions and after encapsulation within the GelMA hydrogel matrix. (Adapted from Lamparelli et al., Biomedical Materials, 2026)

Methodology

PLGA nanocarriers were fabricated via nanoprecipitation using the PreciGenome iFlow™ microfluidic platform equipped with a Y-shaped passive staggered herringbone micromixer (inner diameter 600 µm; total channel length 20 mm). The organic phase consisted of PLGA (Resomer® RG 502 H, 7–17 kDa) dissolved in acetonitrile at 10 mg/mL, while the aqueous phase contained 1% (w/v) poly(vinyl alcohol) and hBMP-2 at 1.33 µg/mL. Operating at a 1:3 oil-to-water flow rate ratio with a total flow rate of 10 mL/min, the platform produced BMP-2–loaded PLGA-NCs with a mean diameter of 144 ± 48 nm, ζ-potential of −21 mV, and encapsulation efficiency of 60%. Batch volume recovery was 96%, demonstrating high reproducibility.

The nanocarriers were incorporated at 1 mg/mL into a 6% (w/v) GelMA hydrogel containing hTSPCs isolated from human semitendinosus tendon. Constructs (3.5 mm diameter × 4 mm height) were bioprinted and photocrosslinked, then cultured under continuous perfusion at 1 mL/min for 21 days using a custom Arduino-controlled peristaltic pump bioreactor.

Figure 2. Physicochemical and morphological characterization of 3D bioprinted scaffolds. (a) FE-SEM images showing scaffold architecture with visible cells and nanocarriers (white circles). (b) Confocal microscopy of rhodamine-labeled PLGA-NCs (red) with DAPI-stained nuclei (blue). (c) Optical microscopy of hTSPCs in 2D culture. (d) Dynamic viscosity of GelMA with and without NCs. (e) Average Feret's diameter from SEM. (Adapted from Lamparelli et al., Biomedical Materials, 2026)

Cell Viability and Dynamic Culture Optimization

Bioprinting preserved high cell viability (approximately 85%), confirming that the PLGA-NC concentration (1 mg/mL) did not substantially increase shear stress during extrusion. Under dynamic perfusion at 1 mL/min, viability recovered to approximately 95% by day 21, with cells exhibiting elongated morphologies consistent with lineage-specific differentiation responses.

Compartmental mathematical modeling demonstrated the superiority of perfusion culture: while glucose diffusion was reasonably effective under static conditions, lactate accumulation was dramatically reduced under dynamic perfusion (concentration difference of 0.2 mol/m³ vs. 1.7 mol/m³ in static culture). This maintained proper sink conditions for BMP-2 release from PLGA-NCs and prevented local pH reduction from PLGA degradation.

Figure 3. Live/dead assay on hTSPCs bioprinted scaffolds cultured under perfusion for 21 days. Live cells stained green (calcein-AM), dead cells stained red (propidium iodide). Viability was approximately 85% immediately after bioprinting and recovered to ~95% by day 21. n = 3 biological replicates. (Adapted from Lamparelli et al., Biomedical Materials, 2026)

Figure 4. Perfusion bioreactor and dynamic compartmental modeling. (a) Schematic of the Arduino-controlled peristaltic pump perfusion system. (b–c) Dynamic modeling showing nutrient and waste distributions under perfusion, demonstrating efficient supply and removal throughout the construct. (Adapted from Lamparelli et al., Biomedical Materials, 2026)

Superior Mineralization in 3D Dynamic Culture

Alizarin Red S staining revealed markedly stronger and progressively increasing calcium deposition in 3D perfused scaffolds compared to conventional 2D static culture with soluble BMP-2 (20 ng/mL). In the 3D dynamic environment, mineral deposition was already evident at days 7 and 14, whereas 2D cultures showed limited staining only at day 21. The advanced mineralization was attributed to the combination of controlled BMP-2 release, maintained sink conditions under perfusion, and the biomimetic 3D microenvironment.

Figure 5. Alizarin Red S staining comparing 2D static culture with soluble BMP-2 (left) versus 3D bioprinted GelMA scaffolds under dynamic perfusion with NC-delivered BMP-2 (right). The 3D dynamic environment promoted significantly earlier and more robust calcium phosphate deposition. (Adapted from Lamparelli et al., Biomedical Materials, 2026)

Enhanced Osteogenic Gene and Protein Expression

Quantitative real-time PCR revealed dramatically enhanced osteogenic gene expression under 3D dynamic conditions. ALP (early osteogenic marker) increased approximately 5-fold in 3D perfused scaffolds versus only ~1-fold in 2D monolayers. Osteopontin (OPN) expression reached 20.9-fold upregulation under 3D dynamic culture compared with 11.7-fold in 2D static conditions (p < 0.05). Osteocalcin (OCN), a late-stage marker, showed an 8-fold increase in 3D culture at days 7 and 21 versus only 4-fold in 2D at day 21. COL1A1 expression increased 4-fold under dynamic conditions but was nearly absent in static culture.

Western blot analysis confirmed these findings at the protein level. In 3D dynamic cultures, OPN expression reached approximately 28-fold by day 21 compared with ~4-fold in 2D cultures (p < 0.05). OCN was consistently detectable with increasing intensity over time in 3D scaffolds, whereas in 2D static cultures it peaked transiently at day 7 and became undetectable thereafter.

Figure 6. Quantitative real-time PCR analysis of osteogenic gene expression in hTSPCs. Markers: ALP, OPN, OCN, COL1A1, COL3A1. Expression normalized to GAPDH, fold change relative to day 0. *p < 0.05; **p < 0.005; ****p < 0.0005. n = 3. (Adapted from Lamparelli et al., Biomedical Materials, 2026)

Figure 7. Western blot analysis and densitometry of osteogenic protein expression. (a) 2D static culture. (b) 3D dynamic perfusion culture. OPN and OCN quantified at days 7, 14, 21. The 3D system exhibited significantly higher and more sustained protein deposition. *p < 0.05; **p < 0.005. n = 3. (Adapted from Lamparelli et al., Biomedical Materials, 2026)

Key Findings at a Glance

Nanocarrier precision via microfluidics — The PreciGenome iFlow™ platform produced uniform 144 nm PLGA nanocarriers with 96% batch recovery and 60% BMP-2 encapsulation efficiency, enabling consistent growth factor delivery.

Rapid, pulsed BMP-2 release — 70% of encapsulated BMP-2 was released within 3 days and 90% by day 6, providing an early osteoinductive pulse that primed hTSPC differentiation without prolonged exposure.

20-fold osteopontin upregulation — hTSPCs in the 3D perfused system showed 20-fold OPN gene expression and 28-fold OPN protein expression by day 21, vastly outperforming conventional 2D static culture.

Dynamic perfusion is critical — Mathematical modeling confirmed that perfusion reduced lactate accumulation 8.5-fold versus static conditions, maintaining optimal sink conditions for NC-mediated release and cell viability.

First 3D osteogenic platform for hTSPCs — This represents the first demonstration of primary human tendon stem cell osteogenic differentiation within a bioprinted, nanocarrier-functionalized 3D microenvironment.

PreciGenome iFlow™ — Featured Formulation Tool

The PreciGenome iFlow™ microfluidic platform was used to fabricate BMP-2–loaded PLGA nanocarriers via nanoprecipitation. Operating with a passive staggered herringbone micromixer (600 µm i.d., 20 mm channel length), the system achieved uniform nanoparticle production at a 1:3 oil-to-water ratio and 10 mL/min total flow rate. The resulting 144 nm nanocarriers with 96% batch volume recovery highlight the platform's exceptional reproducibility and precision for growth factor encapsulation in regenerative medicine applications.

Reference

Lamparelli EP, Lancellotti A, Manzo L, Cortella G, D'Ambrosio A, De Cata D, Piemonte V, Lovecchio J, Maffulli N, Giordano E, Della Porta G. "3D environment with BMP-2-releasing nanocarriers enhances osteogenic commitment of human tendon stem cells." Biomedical Materials. 2026;21(2):025026.

DOI: 10.1088/1748-605X/ae574c

Learn more about PreciGenome iFlow™: www.precigenome.com