Engineering Bioactive Injectable Cements for Enhanced Bone Regeneration: An Immuno-Informed Approach

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Background

Bone is a dynamic and multifunctional organ. Despite its regenerative capacity, approximately 2.2 million orthopedic procedures involving bone grafting and substitution are performed annually worldwide to repair critical defects1.

Limitations of traditional treatments have drawn attention to calcium-phosphate bone cements (CPCs), which resemble the mineral phase of bone. CPC-based bone repair methods rely on pre-formed grafts (blocks or granules), often limited by the complex geometries of defects. Injectable CPCs address this through minimally invasive paste-like delivery and in situ hardening under physiological conditions.

The implantation of biomaterials initiates a foreign body reaction (FBR), in which the innate immune system is the first responder. Macrophages (specialized immune cells) regulate pro-inflammatory/anti-inflammatory responses by recruiting other immune cells to the site16.

Immunomodulation is the ability to modulate this response to promote a favourable microenvironment for tissue repair.

CPCs can be enhanced with bioactive components to modulate the local inflammatory environment.

Manganese (Mn2+), a vital trace element in the human body, supports osteogenic responses through controlled release from the cement matrix to bone microenvironment.

Propolis, a resinous, polyphenol-rich substance collected by bees, offers anti-inflammatory and antioxidant effects3 for immunomodulation and tissue repair.

Purpose

Can a biphasic calcium phosphate bone cement co-delivering propolis-derived polyphenols and Mn2+ ions modulate macrophages toward a pro-regenerative phenotype while maintaining desirable physicochemical properties and exhibiting combined immunomodulatory effects?

Hypothesis

The co-delivery of propolis-derived polyphenols and Mn2+ will induce a measurable shift toward a pro-regenerative macrophage phenotype and maintain the cement’s desired properties at an adequate concentration.

Experimental Design

Four cement formulations were tested:

↳ HA-β-TCP (control)

↳ HA-β-TCP/Mn2+

↳ HA-β-TCP/Propolis

↳ HA-β-TCP/Mn2+/Propolis

Liquid-to-powder ratio (0.5 mL/g) kept constant.

Phase I: Cement Formulation

Biphasic calcium phosphate powder was prepared by homogenizing hydroxyapatite (65 wt%) and β-tricalcium phosphate (35 wt%). MnCO3 (2 wt%) was incorporated as an Mn2+ precursor for controlled ion release during degradation.

Liquid phase consisted of PBS with 1 wt% carboxymethyl cellulose (CMC) to control viscosity. Ethanolic propolis extract (5 v/v%) was incorporated and stirred until homogeneous.

Phases were combined by dropwise addition and spatulation to form a paste, then cast into 6 mm × 3 mm cylindrical moulds.

Phase II: Physicochemical Characterization

Injectability: Assessed via syringe ejection by recording the initial mass of the filled syringe (M0) and residual mass after extrusion (Mr).

Setting time: Time required for a needle to penetrate <1 mm into the cement sample at room temperature.

Degradation: Assessed by recording initial dry weight (Wi) and dry weight after immersion at intervals (Wd).

pH stability: Assessed by immersing samples in 10 mL PBS at pH 7.4 and pH 5.4 (adjusted with HCl). Changes in immersion media were monitored using a digital pH meter.

All experiments were performed in triplicate.

Phase III: Evaluation of Immunomodulation

Pulverized cement was incubated in DMEM for 24 h at 37℃ to produce 10 mg/mL stock solutions. After centrifugation and sterile filtration, six 10-fold serial dilutions were prepared to obtain dose-response data for each treatment.

Human stem cell-derived macrophages were seeded at a density of 6 × 104 cells/well in a 96-well plate and exposed to 100 µL cement-conditioned media for 24 h.

Flow cytometry was used to evaluate macrophage phenotypic markers. Resuspended cells were treated with Fc blocker and stained with antibodies targeting specific surface markers.

Scanning electron microscopy (SEM) was used to examine the surface morphology of granular particles forming aggregated clusters. The least and most concentrated formulations, HA-β-TCP (1, top) and HA-β-TCP/Mn/Propolis (2, bottom), were compared, demonstrating that the incorporation of MnCO₃ and propolis extract did not disrupt the microporous structure of the CPC. (Figure 2)

Phase II: Physicochemical Characterization

Cements demonstrated clinically relevant injectability through various orthopedic cannulas without phase separation. Injectability is highest using a 14-gauge needle (>90%) and decreases as the needle diameter narrows to 21 gauge. (Figure 3)

Adjustment of carboxymethyl cellulose (CMC) concentration largely determined self-setting kinetics. Setting times range from 18 minutes to 32 minutes at room temperature.

Cements exhibited stable weight loss over the 14-day immersion period. The HA-β-TCP/Mn/Propolis formulation demonstrated a higher degradation rate, whereas the HA-β-TCP control showed the lowest, suggesting that the incorporation of Mn2+ and propolis increases the degradation rate, which supports better resorption in vivo. (Figure 4)

When immersed in PBS, pH 7.4, all samples maintained relatively stable pH values over the 15-day immersion period. Media did not exceed the favourable range of pH (7.2-7.6). This shows relevance to in vivo conditions because the extracellular environment is tightly buffered around pH 7.4. A drastic rise from physiological levels can stress or kill cells, impacting immunomodulation. (Figure 5)

Under acidic conditions (PBS, pH 5.4), the cement samples increased local pH over time, indicating buffering activity and ion release from the cement matrix (Ca2+, PO43-, CO3, Mn2+). This shifted the simulated inflammatory environment toward healthy physiological pH levels. (Figure 6)

For percent injectability, differences between all treatments were statistically significant (p < 0.0001) determined by two-way ANOVA with Tukey’s multiple comparisons test.

For degradation and pH stability (pH 7.4 and pH 5.4), differences between the control and HA- β-TCP/Mn, as well as HA- β-TCP/Mn/Propolis, were statistically significant (p < 0.0001), determined by two-way ANOVA with Dunnett’s multiple comparisons test.

Phase III: Evaluation of Immune Modulation

Flow cytometry revealed differences in macrophage CD206 and CD80 expression through

% Expression (Figure 7,8) and Mean Fluorescence Intensity (MFI) (Figure 9,10):

Sample 3, containing propolis alone (HA-β-TCP/Propolis) reaches the highest CD206 MFI at 10 ng/mL, suggesting a dose-dependent effect of propolis on M2 activation that peaks at mid-range doses. The highest %CD206+ observed was an approximate threefold increase from the untreated control within 24 hours.

Sample 2 (HA-β-TCP/Mn) exhibits moderate increases in CD206 MFI and %CD206+ relative to the untreated control, with CD80 expression remaining relatively stable, suggesting that Mn incorporation can enhance M2 polarization without strongly inducing M1 activation.

Sample 4 (HA-β-TCP/Mn/Propolis) maintains higher CD206 MFI across doses. Though there is less clear dose-dependence, CD80 expression was highest at the lowest dose (0.1 ng/mL) and slightly decreased at higher doses, suggesting a combined effect may sustain M2 expression and attenuate M1-associated markers at increased concentrations.

Injectable biphasic calcium phosphate cements were successfully developed and characterized, demonstrating the independent and coordinated delivery of Mn2+ and propolis-derived polyphenols.

All treatments maintained desirable physicochemical behaviour. Injectability through varying orthopedic cannulas offers highly efficient delivery, and cements demonstrate relevant self-setting. Controlled degradation and buffering capacity in vitro suggest a long-term resorption profile that matches the pace of bone regeneration. SEM analysis confirmed that the incorporation of MnCO3 and propolis extract did not disrupt the microporous surface morphology of the cement matrix.

Exposure of stem cell-derived macrophages to cement-conditioned media revealed early immunomodulatory trends, with select treatments promoting a shift toward a pro-regenerative phenotype, as hypothesized. A threefold increase in CD206 expression in propolis-functionalized groups supports further optimization to investigate potential synergistic interactions between polyphenols and bioactive ions.

Results of this preliminary study suggest promising osteogenic potential for future applications in bone tissue repair and regeneration. By incorporating naturally occurring components, the cement provides a biocompatible material suitable for minimally invasive procedures to reduce surgical trauma, risk of infection, and overall treatment costs. As a synthetic CPC matrix enhanced with bioactive additives, it also offers a cost-effective and reproducible alternative to traditional bone grafts while maintaining ideal physicochemical and biological properties (Figure 11).

Moreover, strategies that promote balanced bone homeostasis are increasingly relevant as the global population ages and the prevalence of bone degeneration and fracture-related injuries rises6.  Immuno-informed, bone-mimetic materials like this system can advance regenerative therapies to support effective bone healing even in complex clinical settings.

Future Directions

Systematically vary wt% concentrations of manganese precursor and propolis extract to identify optimal independent and synergistic effects.

Optimize propolis extraction and fractionation to improve yield and bioavailability of phenolic compounds.

Implement simulated body fluid (SBF) immersion media to better replicate physiological conditions and assess in vitro apatite formation.

Quantify ion release and correlate with physicochemical and cellular responses to establish structure-function relationships.

Extend macrophage exposure period (72 h) and introduce inflammatory conditions using lipopolysaccharide (LPS) stimulation.

Further in vitro characterization and progress toward in vivo models are required to evaluate the long-term efficacy and biocompatibility of the scaffold.

I would like to sincerely thank and acknowledge the following individuals and organizations for their support throughout my project:

Swati Joshi and her team at PBR Laboratories for providing laboratory space and technical support during Phases I and II

Dr. Megan Levings and Dr. Bruce Verchere at UBC for connecting me with PhD students Bruno Freitas and Melody Cheng at BC Children's Hospital Research Institute, whose mentorship and expertise in macrophage research during Phase III were invaluable.

Yun Hiebert at SEMx Incorporated for assistance with SEM imaging

My science teacher, Ms. Dawe, for access to school resources

The WBRSF committee for their support and guidance

My parents for their constant encouragement throughout this process

Images and graphics were created using Canva (canva.com) and BioRender (biorender.com), unless otherwise cited. Data and statistics were generated using GraphPad Prism 10 (graphpad.com).

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