Biocompatibility of Nanomaterials in Medical Applications

Marvellous O. Eyube1,2*, Courage Enuesueke1,2, Marvellous Alimikhena1,2

1
Department of Chemistry, Faculty of Physical Science, University of Benin, Nigeria
2
S.T.E.L.L.A.R. Labs (Science and Technological Enhanced Laboratory for Advance Learning and
Research), Benin City, Nigeria
Corresponding Author Email: [email protected]
Corresponding Author ORCID: https://orcid.org/0009-0003-7951-1830

Abstract:

Biocompatibility is a critical factor in the application of nanomaterials in medical fields, as these

materials must interact safely and effectively with biological systems to be viable for therapeutic

and diagnostic use. This article investigates the biocompatibility of nanomaterials, focusing on

their interactions with biological cells, tissues, and the immune system. Key properties such as

surface chemistry, size, shape, and material composition are examined, as they significantly

influence the biological response. The article explores the role of nanomaterials in medical

applications, including drug delivery, diagnostic imaging, and tissue engineering, while discussing

the challenges involved in enhancing their biocompatibility. A case study on the CaO-CaP binary

system is presented, showcasing the use of calcium oxide (CaO) and calcium phosphate (CaP)

nanoparticles in bone tissue engineering. This system is widely investigated for its ability to mimic

the mineral content of bone and promote osteogenesis, highlighting both its therapeutic potential

and challenges in ensuring safe biocompatibility in clinical settings. The article concludes by

reviewing strategies to optimize the biocompatibility of nanomaterials and discussing future

directions for research in advancing their applications in medical treatments.

Keywords: Biocompatibility; CaO-CaP System; Nanomaterials; Tissue-engineering;

Osteointegration; Regeneration
1. Introduction and the Medical Demand for Nanomaterials
Modern medicine is witnessing a paradigm shift, shaped by the rise of precision medicine,

implantable technologies, and patient-specific treatment regimens. These emerging approaches

demand materials that can perform reliably within complex biological systems while enabling fine-

tuned control over therapeutic or diagnostic outcomes. However, conventional biomaterials often

fall short of these requirements. Their limited biological responsiveness, low adaptability, and

potential to trigger immune reactions have created a critical gap in achieving next-generation

medical solutions [1].

Nanomaterials have emerged as transformative candidates capable of addressing these challenges

due to their unique structural and functional properties. Their nanoscale dimensions allow

interaction with biomolecules, cells, and tissues at fundamental biological levels. Features such as

large surface-area-to-volume ratio, tunable surface chemistry, and responsiveness to external

stimuli give them a degree of biofunctionality that conventional materials lack [2]. Furthermore,

their versatility allows them to be integrated into various platforms—from injectable drug delivery

systems to implant coatings—while scalability in production offers potential for widespread

clinical application [3].

These advantages make nanomaterials particularly attractive for real-world medical demands. In

cancer therapy, for example, liposomal formulations such as Doxil® have revolutionized

chemotherapy by delivering doxorubicin directly to tumor sites, reducing systemic toxicity and

improving therapeutic efficacy. This milestone—recognized as the first FDA-approved

nanodrug—demonstrates how rational nanodesign can overcome longstanding limitations in

pharmacokinetics and safety [4]. In diagnostic imaging, ferumoxytol, an iron oxide nanoparticle,
has been successfully used off-label as an MRI contrast agent in various clinical settings,

enhancing vascular imaging in patients for whom conventional gadolinium-based agents are

unsuitable [5]. These examples underscore the ability of nanotechnology to enhance performance

and expand the clinical toolbox across disciplines.

In orthopedic and dental applications, nanostructured coatings and scaffolds—such as those made

from calcium phosphate or calcium oxide—promote bone regeneration and tissue integration due

to their osteoconductive nature [6]. More specifically, nano-hydroxyapatite (nHAp) scaffolds have

shown great promise in clinical and preclinical bone regeneration efforts, offering high surface

reactivity, biomineral mimicry, and superior compatibility with osteoblasts [7]. These scaffolds

not only provide structural support but also serve as bioactive matrices that modulate cell behavior

and promote osteogenesis.

Another critical area of application is tissue engineering, where nanomaterials mimic the

extracellular matrix and support cellular activities necessary for tissue regeneration. These

scaffolds, with high surface area and controllable porosity, facilitate the attachment of cells and

the localized delivery of bioactive agents, enabling the repair of damaged or diseased tissues [8].

Across all these fields, nanomaterials are not only enhancing current medical practices but also

enabling technologies that were previously unachievable with traditional systems.

Despite these promising developments, the clinical use of nanomaterials hinges on their ability to

safely interact with biological environments. Their high reactivity, while beneficial for

functionality, introduces risks of cytotoxicity, inflammation, or immune system activation. As

such, biocompatibility has emerged as a core requirement for successful application in medicine.

Defined as a material’s ability to perform its intended role without provoking adverse biological
responses, biocompatibility ensures that nanomaterials are both effective and safe for clinical use

[1,2].

The next section explores this concept in depth—examining the criteria for assessing

biocompatibility, the mechanisms by which nanomaterials interact with biological systems, and

the strategies employed to mitigate risks. As the foundation for all subsequent medical integration,

biocompatibility forms the critical bridge between nanomaterial innovation and real-world patient

outcomes.

2. The Critical Significance of Biocompatibility in Nanomedicine

2.1. The Role of Biocompatibility in Clinical Success

Biocompatibility is central to the successful application of nanomaterials in medicine. In clinical

settings, it ensures that materials interact with biological tissues without triggering adverse

reactions such as toxicity, inflammation, or immune rejection. Nanomaterials that are not

biocompatible may provoke acute or chronic responses, potentially leading to the failure of a

therapy or device. As medicine increasingly relies on materials that operate at the molecular and

cellular scale, biocompatibility becomes not just desirable, but essential for clinical efficacy and

patient safety [9].

Instances of therapeutic failure due to immune activation, accelerated clearance, or unforeseen

toxicity highlight the need to prioritize biocompatibility early in nanomaterial design. For example,

nanoparticles that perform well under laboratory conditions may behave unpredictably in vivo if

their surface is not tailored to evade immune surveillance or protein fouling. These failures

underscore the importance of understanding how nanomaterials interact with biological systems

in order to reduce the likelihood of clinical setbacks [10].

2.2. Comparison with Conventional Biomaterials

Nanomaterials differ significantly from traditional biomaterials, particularly in their interaction

with biological systems. Unlike bulk materials that are often inert and mechanically focused,

nanoscale materials exhibit high surface energy, increased reactivity, and tunable physicochemical

properties, all of which impact their biocompatibility. These properties enable unprecedented

medical functionalities but also introduce challenges such as protein adsorption, immune

activation, and cytotoxicity if not properly controlled [11].

In contrast to conventional implants made of metals or polymers, nanomaterials are often designed

for more dynamic roles, such as drug delivery or real-time biosensing. Their smaller size enhances

tissue penetration and cellular uptake but also increases the surface area available for interactions

with proteins, lipids, and immune cells. For instance, nanoparticles under 100 nm are efficiently

taken up by cells but may accumulate in tissues or organs, leading to long-term toxicity if they are

not biodegradable [12]. Additionally, particle shape influences behavior—spherical particles are

generally taken up more readily than rod-like ones, which may persist longer and interact

differently with immune pathways [13].

2.3. Regulatory Emphasis on Biocompatibility

Regulatory bodies such as the U.S. Food and Drug Administration (FDA), the European Medicines

Agency (EMA), and the International Organization for Standardization (ISO) have emphasized

biocompatibility as a prerequisite for nanomaterial approval. Beyond proving therapeutic benefit,

developers must provide robust data on toxicity, immunogenicity, biodegradation, and clearance

profiles. These factors are particularly scrutinized for nanomaterials due to their complexity and

the evolving understanding of their interactions with biological systems [14].

Designing for regulatory compliance involves thoughtful material selection and surface

engineering to enhance safety. Organic nanomaterials, for example, often break down into

biocompatible byproducts, making them more favorable for long-term applications. In contrast,

inorganic systems may require surface modifications—such as coating or encapsulation—to meet

safety thresholds and prevent accumulation or chronic toxicity [15,16].

2.4. Foundation for Engineering Nanomedicines

Biocompatibility is not an afterthought—it must be embedded into the nanomaterial’s design from

the start. Surface chemistry, size, shape, charge, and composition all govern how a nanomaterial

interacts with cells and tissues. One common method to improve compatibility is surface

functionalization, such as PEGylation, which extends circulation time and minimizes recognition

by immune cells [10,11]. Surface hydrophilicity is also crucial, as hydrophilic coatings reduce

non-specific protein adsorption and enhance cellular interactions [11].

Surface charge, in particular, plays a dual role. Positively charged nanomaterials can enhance

cellular uptake due to electrostatic interactions with negatively charged membranes but may

simultaneously increase cytotoxicity and inflammation [17]. Negatively charged or neutral

surfaces, though less aggressive in uptake, often present reduced immunogenicity. Achieving a

balanced surface charge is thus critical in optimizing therapeutic performance while minimizing

risks [17].

Another essential parameter is surface energy, which influences how nanomaterials interact with

proteins and form a biological identity—or “protein corona”—upon entering the bloodstream. This

corona can alter distribution, cellular uptake, and immune responses. Modulating surface energy
through chemical design can either suppress or guide these interactions in favor of the intended

application [18].

Material composition further influences biocompatibility. Organic nanomaterials, such as

liposomes and biodegradable polymers, are typically well-tolerated and degrade into safe

byproducts, making them suitable for sustained or repeated administration [14]. On the other hand,

inorganic materials like silica, iron oxide, or gold offer mechanical or imaging advantages but may

require surface modifications to mitigate potential toxicity or long-term accumulation [15]. These

considerations have become particularly important in antiviral nanomedicine, where tailored

surface properties—such as charge, hydrophilicity, and coating strategies—are now central to

ensuring both therapeutic efficacy and immunological safety in viral prevention and treatment

platforms [19].

3. Methodologies for Biocompatibility Assessment

Evaluating the biocompatibility of nanomaterials requires a multidisciplinary approach involving

experimental assays, animal studies, computational modeling, and regulatory frameworks. These

methodologies collectively assess how nanomaterials interact with biological systems, offering a

comprehensive safety and efficacy profile tailored to their medical applications. Given the

complexity of nano–bio interactions, integrating these varied approaches helps researchers

mitigate risks while advancing clinical translation.

3.1. In Vitro Methods

In vitro techniques represent the initial screening tools for assessing the biocompatibility of

nanomaterials. These laboratory-based assays evaluate the cellular responses to nanoparticles

under controlled conditions, eliminating the complexity of whole-organism interactions. Common

methods include cell viability assays such as MTT and resazurin reduction, which quantify

metabolic activity as an indicator of cytotoxicity. Additionally, membrane integrity tests, oxidative

stress assessments, and apoptosis detection are employed to reveal subcellular impacts of

nanomaterials [20].

Real-world applications have demonstrated the efficacy of in vitro tools in nanomedicine research.

For instance, Siller et al. (2019) developed a real-time live-cell imaging system to monitor

cytotoxicity and cellular morphology in response to 3D-printed biomaterials—enabling high-

throughput and time-resolved assessment of biocompatibility [21]. Another case involved the

evaluation of zinc oxide nanoparticles synthesized from Artemisia annua, where MTT assays

confirmed enhanced osteogenic differentiation in human osteoblast-like MG-63 cells [22].

Advanced in vitro systems, including 3D cell cultures and co-culture models, better mimic the

tissue microenvironment compared to traditional 2D monolayers. These systems allow the

observation of nanoparticle-induced changes in cell proliferation, differentiation, and

inflammatory signaling. Although in vitro studies are cost-effective and high-throughput, they lack

the complexity of physiological systems—necessitating follow-up in vivo studies to confirm

findings [20].

3.2. In Vivo Methods

In vivo approaches evaluate biocompatibility within living organisms, providing vital insights into

systemic distribution, metabolism, clearance, and potential toxicity. Rodent models, such as mice

and rats, are commonly used to assess both acute and chronic biological responses. These studies

help identify critical endpoints including immune activation, hematological changes, and organ-

specific toxicities [23].

An exemplary application is the use of CaO–CaP nanocomposites in Wistar rats, where scaffold

implantation in bone defects led to observable tissue regeneration and immune modulation.

Moreover, histopathological examination—central to in vivo assessment—enabled detection of

fibrosis, necrosis, and inflammation across major organ systems. This approach is further

supported by Kyriakides et al. (2021), who provided comprehensive insights into the

immunological outcomes of various nanomaterials in vivo, including cytokine induction,

complement activation, and tissue compatibility [24].

Despite their importance, in vivo methods raise ethical concerns and are limited by species-specific

differences that may not fully predict human responses. Consequently, emerging platforms like

organ-on-a-chip and ex vivo perfusion systems are gaining traction as ethical and functional

alternatives [23].

3.3. Computational Models

Computational models serve as predictive tools for anticipating nanomaterial interactions with

biological systems. Techniques such as molecular dynamics (MD) simulations and quantitative

structure–activity relationship (QSAR) modeling allow researchers to estimate nanoparticle

behavior based on their physicochemical properties. MD simulations help model nanoscale

interactions, such as membrane disruption or protein binding, offering atomic-level resolution of

potential toxicity pathways [25].

Recent developments in nano-QSAR, such as those reported by Cao et al. (2020), have enabled

risk prediction for metal oxide nanoparticles based on computational descriptors like band gap

energy, particle charge, and hydration energy—streamlining early-phase toxicology testing [26].
These models are particularly valuable for screening large nanomaterial libraries without extensive

biological assays.

QSAR models correlate descriptors like particle size, surface charge, and hydrophobicity with

observed biological outcomes. However, their predictive power depends heavily on the quality

and diversity of training datasets. Limitations include the lack of standardized descriptors and

difficulty simulating complex biological microenvironments. Continued refinement and validation

are essential for regulatory acceptance [25,26].

3.4. Regulatory Standards

The regulatory evaluation of nanomaterials is guided by frameworks developed by agencies such

as the U.S. Food and Drug Administration (FDA), European Medicines Agency (EMA), and

international bodies like the International Organization for Standardization (ISO) and the

Organisation for Economic Co-operation and Development (OECD). These organizations mandate

rigorous assessment of toxicity, pharmacokinetics, and biocompatibility prior to clinical approval

[27].

A landmark example of regulatory success is the approval of Doxil®, the first FDA-approved

nanodrug, which underwent extensive in vitro and in vivo biocompatibility testing, including

sterility, hemolysis, and immunogenicity studies [28]. However, current regulatory standards often

lag behind rapid advancements in nanomedicine. Discrepancies in international guidelines and the

lack of harmonized testing protocols remain major barriers to global commercialization.

The regulatory landscape continues to evolve, with agencies pushing for validated in vitro models,

robust in silico predictions, and reliable biocompatibility thresholds. This push for standardization

enhances safety while supporting innovation [27].

3.5. Comparative Metrics and Evaluation Criteria

As multiple assessment techniques are employed, establishing standardized metrics is essential for

comparing biocompatibility outcomes across platforms. Key evaluation criteria include

cytotoxicity thresholds, inflammatory markers (e.g., IL-6, TNF-α), cellular uptake efficiency,

biodistribution profiles, and histological scores. To support objective analysis, benchmark values

are increasingly being established across studies, enabling meta-analyses and cross-comparative

assessments.

Additionally, efforts are underway to create Integrated Testing Strategies (ITS), which combine in

vitro, in vivo, and in silico results into unified decision-making frameworks. These strategies

reduce redundancy, improve predictive accuracy, and accelerate regulatory approval. The concept

is exemplified by projects like Schloemer et al. (2023), who incorporated quantum-based modeling

and biological validation to assess triplet-triplet annihilation in nano-upconversion materials [29].

This convergence of biological and computational evaluation strengthens the translational

potential of nanomaterials. As highlighted by Seoane-Viaño et al. (2021), the successful transition

of 3D-printed drug-delivery systems into clinics depends not just on innovative design but also on

robust biocompatibility evaluation and regulatory navigation [30].

4. Scientific Challenges in Biocompatibility

The rapid integration of nanomaterials into medical science has created opportunities for

breakthroughs in diagnostics, therapy, and regenerative medicine. However, despite their

promising potential, nanomaterials present scientific and translational challenges related to

biocompatibility. These challenges must be addressed to ensure clinical safety, long-term stability,

and regulatory approval. This section outlines the key hurdles faced in biocompatible

nanomedicine, focusing on toxicity, immune interactions, material degradation, and safety

governance.

4.1. Toxicity and Immune Response

One of the primary challenges in deploying nanomaterials for medical use is managing their

toxicological and immunological effects. Nanoparticles, due to their high surface area-to-volume

ratio, can interact intensely with biological components such as proteins, membranes, and immune

cells. While these interactions can be therapeutically useful, they may also provoke unintended

effects such as oxidative stress, pro-inflammatory cytokine release, or complement activation.

Surface properties such as charge, hydrophobicity, and functional groups significantly influence

biocompatibility and immune recognition. For example, positively charged particles may enhance

cellular uptake but are also more likely to disrupt membrane integrity or induce inflammation.

Even particles deemed safe in vitro may exhibit unforeseen toxicity when exposed to the complex

environments of living organisms.

Additionally, the formation of a protein corona on the nanoparticle surface can alter its identity

and behavior in vivo, potentially leading to misrecognition by the immune system. These
complications highlight the need for careful material design and thorough preclinical testing to

mitigate immunotoxic effects.

4.2. Long-Term Stability and Degradation

Nanomaterials intended for biomedical applications must demonstrate stability under

physiological conditions, as well as controlled biodegradation where applicable. Materials that

degrade too quickly may release toxic byproducts, while non-degradable materials may

accumulate in tissues, leading to long-term cytotoxicity or organ burden.

For example, certain inorganic nanoparticles, though stable and effective for imaging or therapy,

may persist in the body without clear metabolic or excretory pathways. This persistence raises

concerns about chronic exposure, particularly for repeated or systemic applications.

Additionally, a major concern with non-biodegradable inorganic nanomaterials is their tendency

to accumulate in vital organs such as the liver, spleen, and kidneys—organs that are central to

clearance and detoxification. Nanoparticles exceeding 50 nm, particularly gold and iron oxide

varieties, often become sequestered in the reticuloendothelial system (RES), where they are

difficult to excrete due to limited renal clearance mechanisms. This long-term accumulation can

lead to chronic inflammation, oxidative stress, or functional impairment in these organs. To

address this challenge, several strategies have emerged: PEGylation of surfaces helps reduce

opsonization and RES uptake; microencapsulation can control release and shield particles from

immune detection; and downsizing nanoparticles below 10 nm enhances renal filtration and

excretion. For example, gold nanoparticles larger than 50 nm show significantly reduced clearance

and prolonged retention in hepatic and splenic tissues, as reported by Kumar et al. (2022) [15].
These solutions are vital for ensuring long-term biocompatibility, especially for nanomaterials

intended for systemic or repeat-dose applications.

Conversely, biodegradable polymers such as polycaprolactone (PCL) and polylactic acid (PLA)

offer a more controlled degradation profile. When engineered appropriately, these materials break

down into non-toxic byproducts, minimizing long-term risk. However, predicting and

standardizing degradation rates across different biological environments remains an ongoing

challenge [31].

4.3. Regulatory and Safety Concerns

The development of biocompatible nanomaterials faces additional hurdles at the regulatory

interface, where evolving safety standards and fragmented global policies can slow innovation.

Regulatory bodies like the FDA, EMA, and ISO have established frameworks for toxicity testing

and biocompatibility assessment, but many of these standards were originally designed for bulk

materials and do not fully account for nano-specific behaviors.

Moreover, safety assessments must now include data on inter-individual variability, long-term

biodistribution, and nanomaterial interaction with complex pathophysiologies, particularly for

personalized and implantable therapies. These requirements demand new testing protocols,

interdisciplinary evaluations, and ongoing collaboration between researchers, industry

stakeholders, and regulatory agencies.

To meet these demands, next-generation nanomedicines must be developed with regulatory

foresight, ensuring that biocompatibility is not only optimized at the design level but also validated

against globally recognized safety benchmarks.

5. Strategies for Targeted Improvement

Optimizing nanomaterials for medical use requires deliberate strategies that address

biocompatibility at the design stage. These strategies not only minimize undesirable biological

interactions but also enhance therapeutic precision, safety, and clinical viability. While many of

these approaches have been extensively validated in laboratory settings, their translation into real-

world systems remains a critical measure of success. This section outlines four core strategies—

surface modification, use of biodegradable platforms, targeted delivery, and hybrid

nanostructures—that lay the foundation for more compatible and functional nanomedical

technologies. These principles are further exemplified in the subsequent section through a focused

analysis of the CaO–CaP binary system.

5.1. Surface Modifications

One of the most effective routes to enhancing biocompatibility is through surface engineering.

Surface modifications, such as PEGylation, have been widely adopted to reduce immune

recognition and prolong nanoparticle circulation time in vivo [3,10,18]. This stealth effect

decreases the likelihood of rapid clearance and allows therapeutic agents more time to reach their

target.

Beyond immune evasion, tuning surface charge, hydrophilicity, and ligand functionalization can

modulate how nanomaterials interact with proteins, membranes, and cells. These alterations help

prevent the formation of a disruptive protein corona, lower immunogenicity, and promote selective

cellular uptake [11,18]. Such modifications are crucial not just in theoretical design but in materials

intended for specific biological environments—as will be later demonstrated in the CaO–CaP

system.
PEGylation, which involves attaching polyethylene glycol (PEG) chains to the nanoparticle

surface, has been one of the most widely used approaches to extend circulation time and evade

immune detection. PEG forms a hydrophilic barrier that resists protein adsorption and

phagocytosis. While effective, PEGylated systems may face clinical challenges such as accelerated

blood clearance upon repeated administration and the development of anti-PEG antibodies.

Ongoing research is therefore focused on optimizing PEG density, architecture, and molecular

weight to maintain efficacy without triggering immune reactions [32].

Zwitterionic surface coatings, composed of molecules bearing both positive and negative charges

(e.g., sulfobetaines, phosphorylcholines), offer an alternative with potentially superior

performance. These coatings create a highly hydrated, non-fouling layer that can completely

suppress protein corona formation—a key factor influencing nanoparticle biodistribution and

immune response. For example, Debayle et al. demonstrated that zwitterionic ligands effectively

eliminated protein adsorption, outperforming conventional PEGylation in stability and stealth

behavior under physiological conditions [33].

Another promising immune evasion approach involves biomimicking natural “do-not-eat-me”

signals. The most studied of these is the surface presentation of CD47 peptides, which interact

with the signal regulatory protein alpha (SIRPα) receptor on macrophages to inhibit phagocytosis.

Mimicking this immune checkpoint mechanism allows nanoparticles to circulate longer and avoid

premature clearance. However, challenges related to hematological toxicity and over-suppression

of immune surveillance must be carefully managed in therapeutic applications [34].

Finally, biomimetic membrane coatings have emerged as a multifunctional platform that enhances

biocompatibility, targeting, and systemic stability. In this approach, nanoparticles are cloaked with
membranes derived from red blood cells, platelets, leukocytes, or even cancer cells, allowing them

to evade immune detection and exhibit tissue-specific homing capabilities. These membrane-

coated nanocarriers possess native surface proteins and antigens, facilitating immune

camouflaging and prolonged blood residence. Recent studies have shown their potential in drug

delivery, detoxification, and vaccine delivery systems, reinforcing their role as next-generation

bioinspired vehicles [35].

Together, these advanced surface engineering strategies represent a critical arsenal for designing

nanomaterials that can navigate the complex immune landscape of the human body, enhancing

both safety and efficacy in clinical applications.

5.2. Biodegradable Nanomaterials

Biodegradable nanomaterials, particularly those based on natural or synthetic polymers like

polycaprolactone (PCL) and polylactic acid (PLA), offer intrinsic compatibility with biological

systems [14]. These materials gradually degrade into non-toxic byproducts, which reduces the risk

of long-term accumulation and associated chronic toxicity.

Crucially, the degradation profile of these materials can be engineered to match therapeutic

timelines, enabling sustained or controlled release of active agents. This feature is especially

relevant in regenerative medicine and drug delivery, where time-sensitive release and safe

clearance are critical for success [10,14]. The use of such materials, as seen in composites like

CaO–CaP, reflects the importance of selecting biodegradable constituents in real-world

applications.
5.3. Targeted Delivery

Targeting strategies have revolutionized the precision of nanomedicine. By conjugating

nanocarriers with ligands specific to disease markers—such as overexpressed receptors in

tumors—researchers can enhance drug accumulation at the intended site while limiting systemic

exposure [10]. Furthermore, the development of stimuli-responsive systems, which respond to

local triggers like pH or enzymatic activity, allows for context-sensitive drug release. These smart

delivery platforms reduce collateral tissue damage and improve therapeutic outcomes, making

them ideal candidates for diseases that require localized treatment such as cancer [10,31].

Recent advancements have highlighted the role of such nanocarrier systems in colorectal cancer

management, where multifunctional nanomaterials have been employed for simultaneous

diagnosis, targeted drug delivery, and therapeutic monitoring. These systems are designed to

selectively accumulate in tumor sites, enhancing treatment specificity while minimizing off-target

effects—offering a practical model of site-specific therapy that aligns with biocompatibility and

translational requirements [36]. Importantly, materials designed for such systems—including

calcium-based nanocarriers—must be engineered with biocompatibility in mind, as explored in

the next section.

5.4. Hybrid Systems

Hybrid nanostructures, which integrate organic and inorganic components, offer unique

opportunities to combine functionality with biocompatibility. For instance, metallic cores like gold

or calcium compounds can be coated with biodegradable or bioactive polymers, balancing

structural stability with reduced toxicity [3,15].

These systems are particularly suited for theranostics, where a single nanomaterial performs both

diagnostic and therapeutic roles. However, the integration of multiple material types necessitates

careful control over surface chemistry, charge, and degradation behavior to maintain biological

harmony [3,18]. The CaO–CaP binary system, discussed next, represents a hybrid platform that

exemplifies these design principles in a biomedical context.

In addition, while these strategies provide the conceptual foundation for improving

biocompatibility, their effectiveness must be validated through application-specific testing. In the

following section, we turn our attention to a case-based empirical analysis of the CaO–CaP binary

system, showcasing how these theoretical strategies are implemented and evaluated in a real-world

biomedical scenario.

6. Case-Based Empirical Analysis: CaO-CaP Binary System

The successful translation of nanomaterials into clinical applications relies on their ability to

balance functional performance with biocompatibility. As discussed in the previous section,

targeted strategies such as surface modification, biodegradability, and composite design are

foundational. The CaO–CaP binary system provides a compelling case study in this regard,

illustrating both the promise and the challenges of deploying biocompatible nanomaterials in

regenerative medicine. Based on empirical work and laboratory experience, this section explores

the key features, in vitro and in vivo findings, clinical challenges, and future strategies associated

with CaO–CaP nanocomposites in bone tissue engineering.

6.1. Material Properties and Molecular Mechanisms

The binary system comprising calcium oxide (CaO) and calcium phosphate (CaP) leverages the

individual strengths of both materials. CaO is known for its high alkalinity and rapid dissolution,

facilitating a bioactive environment that promotes mineralization and bone induction. CaP, being

structurally similar to the mineral phase of bone, contributes long-term mechanical stability and

degradation. The material properties and molecular mechanisms of the CaO-CaP nanomaterial are

illustrated. As shown in Figure 1, the structural and functional attributes of the CaO–CaP

nanomaterial are closely linked to its molecular interactions and phase composition.

Figure 1. Diagram illustrating the material properties and molecular mechanisms of the CaO-CaP
Nanomaterial.
The synergy between these materials lies in their complementary degradation kinetics and ion

release. CaO initiates early-stage mineral deposition by releasing calcium ions, while CaP

maintains a scaffold architecture that supports prolonged cell adhesion and tissue integration. This

dual-phase release profile fosters hydroxyapatite formation and enhances scaffold–tissue

interactions, aligning with broader trends in calcium phosphate-based material design for

osteogenic applications [37,38,39].

The synergistic interplay between calcium oxide and calcium phosphate in composite scaffolds is

central to their osteoconductive and structural functions. Recent literature has emphasized that

integrating other bioactive ions, such as magnesium, further augments these properties. For

example, Qi et al. demonstrated that magnesium-containing bioceramics not only improved

scaffold bioactivity but also enhanced osteoblast function and angiogenesis—two vital processes

for successful bone regeneration. These findings suggest that controlled ion release, whether from

Ca²⁺, PO₄³⁻, or Mg²⁺ sources, creates a favorable microenvironment that mimics natural bone

remodeling, thus reinforcing the functional design principles employed in CaO–CaP systems [40].

In addition to promoting bone regeneration, CaO–CaP scaffolds exhibit promising antimicrobial

properties, which are increasingly valued in preventing implant-associated infections. The

antimicrobial mechanism primarily arises from CaO’s high alkalinity, which results in localized

pH elevation upon dissolution. This shift in microenvironment disrupts bacterial membrane

integrity, denatures proteins, and inhibits enzymatic activity, ultimately leading to bacterial cell

death [41].

Studies have demonstrated that the inclusion of calcium-based nanoparticles—such as CaO and

CaO₂—can significantly reduce microbial viability in wound healing and bone repair contexts. For
instance, Yu et al. (2023) showed that sprayed PAA-CaO₂ nanoparticles enhanced wound healing

through a synergistic release of calcium ions and reactive oxygen species, effectively suppressing

bacterial proliferation [42]. Similarly, Levingstone et al. (2019) confirmed that calcium phosphate-

based scaffolds supported osteogenesis while displaying resistance to microbial colonization in

vitro [43].

Although published studies on CaO–CaP systems are still emerging, ongoing investigations at

S.T.E.L.L.A.R LABS are exploring their efficacy against Staphylococcus aureus, a common

pathogen in orthopedic infections. Early in vitro findings indicate reduced bacterial adherence and

enhanced scaffold sterility—suggesting the potential for dual-functionality: facilitating bone

regeneration while mitigating infection risk. This integrated therapeutic approach reflects the next

frontier in regenerative biomaterials—designing scaffolds that both heal and protect.

6.2. Biocompatibility Studies (In Vitro and In Vivo)

Extensive in vitro and in vivo assessments of CaO–CaP nanomaterials have underscored their

favorable biological interactions. In vitro assays conducted in osteoblast cell cultures demonstrated

robust cell attachment, proliferation, and differentiation. Scaffolds exhibited minimal cytotoxicity,

and the controlled release of calcium and phosphate ions effectively promoted mineralized matrix

formation [44].

These in vitro results were corroborated by in vivo experiments using rodent models, where CaO–

CaP scaffolds were implanted into critical-sized bone defects. Histological analyses revealed

successful osseointegration, dense bone ingrowth, and strong interfacial bonding between the

scaffold and host tissue. In comparative studies, CaO–CaP outperformed conventional grafts in
promoting bone regeneration and defect closure, highlighting its bioactivity and compatibility [45,

46].

6.3. Clinical Bottlenecks and Inflammation Response

Despite its potential, the CaO–CaP system presents notable challenges in clinical translation—

chief among them being its degradation-related inflammation. The rapid dissolution of CaO can

lead to elevated local calcium ion concentrations and alkaline pH, triggering inflammatory

responses in surrounding tissue.

Our own observations in preclinical models confirmed this issue: regions with accelerated scaffold

degradation exhibited localized inflammation and mild immune activation. These reactions were

likely mediated by macrophage response to pH shifts and ion overload, particularly in early post-

implantation stages.

To mitigate this, surface coatings have proven effective. Biodegradable polymers such as

poly(lactic-co-glycolic acid) (PLGA) and polyethylene glycol (PEG) were applied to modulate ion

release and reduce pH-related stress. These coatings resulted in delayed degradation, lower

inflammatory response, and preserved scaffold bioactivity—findings that align with prior studies

on CaO composites and surface-functionalized bone scaffolds [47,48]. As shown in Figure 2,

coating CaO–CaP with PLGA initiates a pH-regulated sequence of reactions that ultimately

influences the release of pro-inflammatory cytokines.

Figure 2. Diagram illustrating how the surface modification of CaO-CaP (such as adding PLGA
coating) triggers a series of reaction by adjusting pH and ultimately affects the secretion of pro-
inflammatory cytokines.

Further analysis of the inflammatory microenvironment revealed that early-stage responses (within

0–7 days post-implantation) were characterized by elevated levels of pro-inflammatory cytokines

such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and interleukin-1 beta (IL-1β).

These mediators contribute to tissue swelling, leukocyte recruitment, and vascular changes. In the

subacute phase, the inflammatory signal begins to subside, making way for reparative processes.

A critical aspect of recovery is the phenotypic transition of macrophages from a classically

activated (M1) to an alternatively activated (M2) state. This shift is associated with increased

secretion of anti-inflammatory cytokines like interleukin-10 (IL-10), which downregulate the

immune response and promote tissue regeneration. Modulating this immune balance through

material design and surface treatment is therefore key to minimizing long-term tissue damage and

enhancing scaffold integration. As shown in Figures 3 and 4, PLGA modification alters the

functional behavior of CaO–CaP nanomaterials, while subsequent scaffold degradation triggers a

time-dependent inflammatory response marked by shifts in cytokine expression.

 Tissue Irritation Pro-inflammatory
and Immune Cytokines (e.g.,
Activation IL-6, TNF- α
Rapid Ca2+
Release and
CaO-CaP Scaffold Alkaline pH

Controlled Ion Stable pH Inflammation and

Release Environment Cytokinine
PLGA Coated Secretion
CaO-CaP

Figure 3. Diagram illustrating how PLGA modifies the behavior of CaO-CaP Nanomaterials.

NB: -

The uncoated pathway shows rapid calcium ion release and pH elevation, leading to tissue
irritation and the upregulation of inflammatory cytokines like IL-6 and TNF-α.

The PLGA-coated pathway moderates’ ion release and stabilizes pH, resulting in reduced
inflammation and improved biocompatibility.
Figure 4. Inflammation Timeline and Cytokine Dynamics Following CaO–CaP Scaffold
Degradation

6.4. Emerging Strategies for Clinical Translation

Translating CaO–CaP systems into widespread clinical use requires not only addressing biological

performance but also scaling design innovation. Current advancements include the incorporation

of osteogenic growth factors, such as BMPs and VEGF, which enhance vascularization and

osteoblast activity [49]. Additionally, gene-activated scaffolds that deliver DNA or RNA

sequences directly to regenerative sites are showing promise in treating complex or non-healing

bone defects.

Another frontier is 3D printing, which enables the fabrication of patient-specific scaffolds that

precisely match anatomical geometries. This personalization improves implant fit, mechanical
loading distribution, and healing outcomes. Our ongoing research supports these trends, with CaO–

CaP scaffolds being explored in combination with bioactive molecules and additive manufacturing

technologies to enhance both regenerative efficacy and clinical adaptability [50].

In parallel with these developments, magnesium-doped biodegradable systems are gaining traction

as promising platforms for enhanced bone regeneration. Recent findings by Tao et al. [51]

highlight the successful fabrication of porous polylactic acid (PLA) microspheres integrated with

magnesium ions, which demonstrated improved biocompatibility, osteogenic activity, and scaffold

resorption dynamics. These outcomes align with our ongoing investigation into CaO–CaP systems,

where controlled ionic release and structural adaptability are key. The supportive role of

magnesium in modulating cellular responses and promoting bone matrix formation underscores

the relevance of ion-enhanced strategies for optimizing the clinical translation of CaO–CaP-based

therapies. As shown in Tables 1 and 2, selected nanomaterials exhibit varying degrees of

biocompatibility across different evaluation criteria.

Table 1: Comparative Biocompatibility Parameters of Selected Nanomaterials

Nanomaterial Hemolysis Complement Circulation Cytotoxicity Remarks

Rate Activation Half-life Level
Gold Low (<5%) Moderate Moderate Low Excellent
Nanoparticles (dose- (~24 h) imaging agent;
(AuNPs) dependent) surface-
dependent
immunogenicity
Silica Moderate High (due to Short (<12 Moderate High surface
Nanoparticles (10–15%) surface h) reactivity;
(SiNPs) silanol) surface
passivation
 improves
compatibility
Lipid Very Low Minimal Long (up to Low Used in mRNA
Nanoparticles (<2%) several vaccines; highly
(LNPs) days) biocompatible
Calcium Low (<5%) Minimal Biodegrada Very Low Excellent for
Phosphate ble bone integration
(CaP) and
mineralization
Calcium High Moderate to Fast- High Requires coating
Oxide (CaO) (>15%) high degrading (alkalinity- to reduce
uncoated induced) cytotoxicity
(e.g., PLGA,
PEG)
PLGA-Coated Low (<3%) Low Controlled Very Low Reduced
CaO–CaP (tailored by inflammation
design) and enhanced
osteointegration
Carbon Variable High (can Long (>48 Moderate to Requires
Nanotubes (depends on activate h) High functionalization
(CNTs) type) immune to improve
cells) compatibility
Quantum High High Long (up to High Toxic elements
Dots (QDs) (>20%) several (e.g., Cd);
days) limited clinical
use without
shielding
strategies
Table 2: Comparative Biocompatibility Metrics of Selected Nanomaterials

Nanomaterial Hemolysis Complement Circulation Inflammatory

Rate (%) Activation Half-life Response (IL-

(C3a Level) (hours) 6/IL-1β)

CaO–CaP 4.5 Moderate 6–8 Low (with

coating)

Gold 2.1 Low 12–24 Minimal

Nanoparticles

Silica 7.8 High 2–5 Elevated

Nanoparticles

PLGA 3.3 Low 8–12 Minimal

Nanoparticles

Lipid 1.2 Very Low 24+ Negligible

Nanoparticles

In pursuit of improved therapeutic outcomes, scaffold systems are increasingly being

functionalized with biologically active molecules. Among these, growth factors such as bone

morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) have shown

particular promise for bone regeneration and angiogenesis [64]. These biomolecules are often

delivered using biodegradable carriers such as PLGA microparticles, which provide sustained

release and localized bioactivity. Additionally, scaffold-mediated gene delivery approaches have

gained traction—particularly with plasmid DNA encoding VEGF or BMP-2 being immobilized

within calcium phosphate-based (CaP) scaffolds. These gene-activated matrices promote

prolonged expression of regenerative signals directly at the defect site. However, this strategy is

not without challenges. Maintaining vector stability during fabrication, achieving dose-controlled

transfection, and avoiding off-target gene expression remain significant barriers to clinical

translation.

Furthermore, additive manufacturing techniques have opened new possibilities for personalized

scaffold design. For instance, in 2022, patient-specific craniofacial bone scaffolds printed with

calcium phosphate achieved a 95% clinical success rate, underscoring the feasibility and efficacy

of tailored regenerative implants [49]. Technologies such as fused deposition modeling (FDM)

and stereolithography (SLA) are now being used to fabricate constructs that mirror patient-specific

anatomical geometries. These 3D printing approaches not only enhance structural fit but also

enable spatial control over porosity, mechanical properties, and bioactive component

distribution—features that are difficult to achieve with traditional fabrication methods. As shown

in Figure 5, bone tissue engineering involves coordinated approaches that enhance regeneration

through scaffold design, signaling cues, and bioactive materials.

Figure 5. Diagram illustrating the synergistic strategies in bone tissue engineering

The CaO–CaP binary system exemplifies the delicate balance between biological reactivity and

structural stability that defines successful nanomaterial applications in medicine. While its

osteogenic potential and favorable integration are well-established, fine-tuning degradation rates

and immune compatibility remains crucial. As ongoing innovations in surface coatings,

biofunctionalization, and manufacturing techniques evolve, CaO–CaP composites stand poised for

greater clinical relevance, offering a tangible example of how theoretical biocompatibility

strategies can translate into real-world biomedical impact.

7. Emerging Trends and Future Directions in Nanomedicine

The trajectory of nanomedicine continues to evolve through technological convergence and

multidisciplinary innovation. Advanced nanomaterials—such as quantum dots, carbon-based

nanostructures, and multifunctional hybrid platforms—are at the forefront of this revolution. These

next-generation materials offer enhanced optical, electrical, and mechanical properties, alongside

superior biocompatibility, making them ideal for applications in targeted diagnostics, image-

guided therapy, and precision drug delivery [53,53].

Recent advances have further demonstrated the clinical promise of nanotechnology in diverse

therapeutic areas. For instance, multifunctional nanoplatforms have been successfully applied in

colorectal cancer management—supporting integrated roles in early screening, targeted drug

delivery, and image-guided treatment, thereby offering a synergistic approach to diagnosis and

therapy [54]. Similarly, engineered nanomaterials have shown considerable effectiveness in the

prevention and treatment of viral infections, particularly through surface modifications that

enhance immune compatibility and targeted delivery of antiviral agents [19].

To address scalability and quality control challenges in nanomaterial production, several

innovative approaches have been proposed and implemented. For instance, researchers at the

University of Liverpool’s Centre for Regulatory Nanomedicine (Smith & Taylor’s group)

overcame nanoparticle aggregation during scale-up by employing spray-drying techniques in place

of traditional solvent evaporation, significantly enhancing batch uniformity and yield [55].

Additionally, automated microfluidic platforms are being explored to enable continuous,

reproducible nanoparticle synthesis with real-time process control—reducing inter-batch

variability and improving scalability. Also, at our lab—S.T.E.L.L.A.R. LABS, current efforts are
being focused on integrating AI-guided microfluidic synthesis and in-line spectroscopic

monitoring to standardize particle size, morphology, and functionalization across production

batches. These advancements are essential to ensure clinical-grade reproducibility and regulatory

compliance in future nanomedicine applications.

In tandem, artificial intelligence (AI) and machine learning (ML) are redefining how nanomaterials

are designed, synthesized, and validated. AI-enabled modeling accelerates the optimization of

physicochemical properties while predicting biocompatibility outcomes with greater accuracy.

These computational approaches support rational design and reduce experimental costs, helping

streamline preclinical development and safety assessment [56].

Another transformative shift is the expansion of personalized medicine, where nanotechnology

plays a pivotal role in tailoring treatments based on individual genetic and physiological profiles.

One of the most prominent success stories is the application of lipid nanoparticles (LNPs) in

mRNA vaccine delivery, as seen in the COVID-19 pandemic. LNPs serve as efficient carriers

capable of protecting nucleic acids and delivering them to specific target cells while minimizing

systemic side effects. This model now informs a broader wave of therapeutic strategies for

oncology, genetic disorders, and rare diseases [57]. As demonstrated in recent studies, the ability

of nanocarriers to act both as immune modulators and targeted therapeutic vectors positions them

as crucial tools in fighting infectious diseases and complex cancers alike [19,58].

Despite these breakthroughs, the field must continue to address significant regulatory and

translational hurdles. The dynamic nature of nanomaterials often outpaces the capabilities of

traditional regulatory frameworks. To bridge this gap, global organizations such as the OECD and

collaborative consortia have intensified efforts to harmonize testing protocols, standardize safety
evaluations, and develop guidelines for clinical translation. Progress in these areas, particularly

through public–private partnerships and international cooperation, is gradually reducing

translational bottlenecks [60,61].

As the discipline advances, it becomes increasingly clear that the future of nanomedicine lies in

interdisciplinary fusion: combining smart material science, computational biology, regulatory

foresight, and patient-specific therapy. This integrated vision not only strengthens the scientific

foundation of nanomedicine but also accelerates its real-world impact in improving global health

outcomes.

8. Conclusion

Biocompatibility remains a foundational requirement for the effective use of nanomaterials in

medical fields such as drug delivery, diagnostic imaging, tissue regeneration, and antimicrobial

therapy. Key parameters—including surface chemistry, particle size, and material composition—

critically determine biological responses, as illustrated by the promising performance of the

CaO–CaP binary system in bone tissue engineering. The complementary properties of calcium

oxide and calcium phosphate, when combined with surface modifications, demonstrate strong

potential for clinical osteointegration.

As the field progresses, innovations in materials science and cross-disciplinary collaboration are

expected to overcome persistent challenges related to toxicity, immune compatibility, and large-

scale application. The integration of computational tools and artificial intelligence will further

streamline the design and prediction of safer, high-performance nanomaterials. Although

regulatory complexities continue to pose barriers, coordinated efforts among scientists,

clinicians, and regulatory bodies will be vital in driving successful clinical translation. By
centering biocompatibility in design and addressing translational gaps, nanomedicine is poised to

reshape modern healthcare and unlock solutions once beyond reach.

Author Contributions

 Marvellous O. Eyube: Led the research project and coordinated all activities. He wrote
Sections 1 (Introduction and the Medical Demand for Nanomaterials), 4 (Medical
Applications of Biocompatible Materials), 6 (Case-Based Empirical Analysis: CaO–
CaP Binary System), 7 (Emerging Trends and Future Directions), and 8 (Conclusion).
He also handled overall manuscript supervision, critical revision, and final approval.
 Courage Enuesueke: Conducted laboratory investigations and data analysis for the CaO–
CaP system. Authored Sections 2 (The Critical Significance of Biocompatibility in
Nanomedicine) and 4 (Scientific Challenges in Biocompatibility). Also contributed to
content review, formatting, and scientific accuracy.
 Marvellous Alimikhena: Wrote Sections 3 (Methods of Assessing Biocompatibility)
and 5 (Strategies for Targeted Improvement). Contributed to figure preparation,
reference formatting, and proofreading.

All authors contributed to the conceptual development and approved the final version of the
manuscript.

Acknowledgments

First and foremost, the authors give all glory to God Almighty for the wisdom, strength, and grace
to complete this research.

We gratefully acknowledge STELLAR Labs for their invaluable research support, mentorship,
and access to laboratory resources that made this work possible. We also extend our appreciation
to the Department of Chemistry, Faculty of Physical Sciences, University of Benin, for
providing the infrastructure and technical assistance necessary for the successful execution of this
study.

Special thanks go to the technical staff for their help during the experimental phases and to
colleagues who contributed through discussions and feedback.
A heartfelt acknowledgment goes to Marvellous O. Eyube for his outstanding leadership,
guidance, and commitment throughout the project.

Appendix

A1. Experimental Methods for CaO–CaP Composite Synthesis

 Materials Used:
o Snail shells (as the natural source of calcium carbonate, CaCO₃)
o Sodium dihydrogen orthophosphate (NaH₂PO₄)
o Ascorbic acid
o Deionized water
o Ethanol (EtOH)
 Synthesis Process:
1. Preparation of CaO:
 Cleaned snail shells were dried and then calcined in a muffle furnace at
900–950°C for 3 hours to convert CaCO₃ into CaO.
 The resulting white CaO powder was ground and sieved for uniformity.
2. Synthesis of Calcium Phosphate (CaP):
 A solution of sodium dihydrogen orthophosphate was prepared and
slowly added to a CaO suspension in deionized water under constant
stirring.
 Ascorbic acid was used as a stabilizing and pH-regulating agent during the
reaction.
 The reaction mixture was stirred for several hours at room temperature and
then aged for 24 hours.
3. Composite Formation:
 The precipitate was filtered, washed with ethanol and deionized water,
and then dried at 80°C.
 Dried powder was ground and calcined at 600°C to enhance crystallinity.
 The final CaO–CaP binary composite was formed by blending CaO and
CaP in various ratios (e.g., 90:10, 85:15).

A2. Cell Culture Protocol (In Vitro Biocompatibility Testing)

 Cell Line: Human osteoblast-like cells (MG-63)

 Medium: DMEM with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin
 Procedure:
o Cells were seeded on CaO–CaP scaffolds and incubated for 1–7 days.
o Cell viability was measured using MTT assay.
o Morphological assessment was done via scanning electron microscopy (SEM) post-
fixation and dehydration.

A3. In Vivo Animal Model Summary

 Animal Model: Wistar rats (n = 12), study approved by the Institutional Animal Care and
Use Committee (IACUC).
 Procedure:
o A critical-sized bone defect (~5 mm) was created in the rat calvaria.
o CaO–CaP scaffolds were implanted; controls included defect-only and CaP-only
groups.
o Rats were monitored for 4, 8, and 12 weeks.
 Assessment Techniques:
o Histological analysis for inflammation and bone regeneration
o Micro-CT imaging for structural integration
o Blood analysis for inflammatory markers
A4. Table Descriptions

Table 1: Comparative Biocompatibility Parameters of Selected Nanomaterials

This table presents key biocompatibility indicators—such as hemolysis rate, complement

activation level, half-life, and cytotoxicity—across a selection of commonly used nanomaterials.
Materials covered include gold nanoparticles (AuNPs), lipid nanoparticles (LNPs), carbon
nanotubes (CNTs), and CaO–CaP composites. The table enables direct comparison of biological
safety profiles to guide material selection in biomedical applications.

Table 2: Summary of Biocompatibility Evaluation Methods and Metrics

An overview of standard in vitro, in vivo, and in silico techniques used to assess nanomaterial
biocompatibility. The table outlines corresponding evaluation criteria (e.g., cell viability,
histological scoring, QSAR modeling parameters) and highlights typical application scenarios. It
supports Section 3 by contextualizing methodological strengths and limitations across different
testing platforms.

A4. Figure Descriptions

Figure 1: Schematic of CaO–CaP Scaffold Degradation and Ion Release

This figure illustrates the dissolution behavior of CaO–CaP composites in physiological

environments. Upon implantation, the scaffold releases Ca²⁺, PO₄³⁻, and OH⁻ ions, leading to
changes in local ionic concentration and pH. These physicochemical changes influence both
bone regeneration and immune responses, highlighting the importance of controlled degradation
rates.

Figure 2: Inflammatory Pathways Triggered by CaO–Induced pH Elevation

A molecular schematic showing how the alkaline environment produced by CaO dissolution
activates inflammatory signaling. The diagram includes macrophage recruitment, cytokine (IL-6,
TNF-α, IL-1β) release, and downstream immune activation pathways. This cascade represents a
major bottleneck in the clinical translation of unmodified CaO-based scaffolds.

Figure 3: Effect of Surface Coatings on Scaffold Immunomodulation

This diagram compares uncoated and polymer-coated CaO–CaP scaffolds (e.g., PLGA or PEG).
It shows how surface engineering delays ion release, stabilizes pH, and reduces macrophage
activation. The figure includes data-supported annotations referencing cytokine levels and
immune cell profiles, based on in vitro and in vivo studies.

Figure 4: Inflammation Timeline and Cytokine Dynamics Following CaO–CaP

Scaffold Degradation

A time-based chart depicting acute (0–7 days), subacute, and recovery phases of inflammation.
The y-axis represents cytokine levels (e.g., IL-6, TNF-α, IL-1β, IL-10), and the curve shows the
rise and resolution of inflammatory markers. The figure also maps macrophage polarization from
M1 to M2 phenotype, underscoring the immune modulation over time post-implantation.

Figure 5: Synergistic Strategies in Bone Tissue Engineering

(a) Gene-activated scaffold delivering therapeutic genes (e.g., VEGF, BMP-2) via plasmid DNA
or controlled release from PLGA microparticles, promoting localized osteogenesis and
angiogenesis.
(b) Additive manufacturing techniques such as fused deposition modeling (FDM) and
stereolithography (SLA) used to fabricate patient-specific scaffolds with biomimetic geometry
and tunable porosity.
The figure highlights how the integration of biofunctionality and structural precision enables
next-generation scaffold development.

Note: Raw experimental data and SEM images are available upon request or can be submitted as
supplementary material.
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