A search for publication data on in vivo studies of HAp was performed on September 5, 2025, using the Scopus database. Scopus was selected as the principal database for acquiring peer-reviewed publications because of its extensive coverage, sophisticated analytical features, and rigorous selection criteria. Moreover, Scopus provides extensive citation metrics, including weighted field citations, which aid in assessing the impact of certain publications and journals. ^{16, 17} This quantitative data is essential for scholars aiming to discern the most impactful literature in their fields. Consequently, Scopus offers numerous advantages over Google Scholar and Web of Science, establishing it as the preferred option for researchers pursuing comprehensive bibliometric data. The steps for document retrieval are shown in Figure 1.

The initial phase entailed an identification process, during which an article search was performed in the Scopus database utilizing the keywords: in vivo study AND hydroxyapatite, focusing on the article title, abstract, and keyword section. The resultant documents totaled 6,353 records. Subsequently, article screening was conducted using the specific keywords: “in vivo study” AND “hydroxyapatite”. The specified keywords yielded 2,876 documents in total. The search for papers was subsequently restricted to those published between 2015 and 2025. Furthermore, document types including reviews, conference papers, book chapters, retracted editorials, letters, conference reviews, short surveys, notes, and errata were excluded. Consequently, 1704 eligible articles were retrieved. Additionally, articles in Chinese, Russian, Czech, Portuguese, and Ukrainian were excluded, resulting in the exclusive use of texts written in English. A total of 1,682 studies were included in the review.

In this study, a bibliometric analysis was performed using VOSviewer. Co-authorship analysis was applied to map collaborative networks among authors, institutions, and countries, highlighting the scientific partnerships that drive research development in this field. Co-citation analysis was conducted to reveal the intellectual structure of the domain by identifying journals and authors most frequently cited together, reflecting the theoretical foundations underpinning HAp research. Bibliographic coupling analysis was employed to assess similarities among documents, institutions, or countries based on shared references, which helped uncover research directions and thematic linkages across scientific entities. Finally, keyword co-occurrence analysis was used to map thematic clusters and emerging research trends, particularly in relation to scaffolds, osseointegration, ionic doping, and composite applications in in vivo HAp studies. ^{18– 22}

The conclusions of this study are based on the findings of 1,682 papers sourced from the Scopus database regarding in vivo investigations of HAp. Research from 2015 to 2025 indicated an upward trend until 2021 (186 documents), followed by a decline in 2022 (155 documents). Research escalated once more until 2024, yielding 176 documents, and then declined in 2025, resulting in 119 documents. This indicates that research on in vivo investigations of HAp remains limited, suggesting that it is a viable area for future studies ( Figure 2).

Analysis of international collaboration, visualized through VOSviewer using bibliographic coupling and focusing on countries, revealed that research publications were predominantly authored by China (704 documents), followed by the United States (245 documents), India (118 documents), South Korea (109 documents), Germany (83 documents), Japan (82 documents), Brazil (73 documents), Iran (68 documents), Italy (60 documents), and the United Kingdom (60 documents), as illustrated in Figure 3.

In recent years, research publications have been predominantly authored by the United Arab Emirates (2 documents) in 2025, Croatia (2 documents) in 2025, Iraq (3 documents) in 2023, Thailand (11 documents) in 2022, Colombia (5 documents) in 2022, Vietnam (4 documents) in 2022, and Tunisia (3 documents) in 2022. The findings indicate that the international research network focused on in vivo studies of HAp is expanding, resulting in new investigations in various nations. Research collaboration with China has yielded significant contributions from various countries, including the United Arab Emirates, Vietnam, Thailand, Iraq, Bosnia and Herzegovina, Ukraine, Colombia, and Croatia. Research collaboration with the United States yielded significant contributions from various countries, including Tunisia, Iraq, Vietnam, Thailand, Bosnia and Herzegovina, Croatia, and Colombia. Research collaboration with India has yielded significant contributions from several countries, including Vietnam, Thailand, and Colombia. Furthermore, this finding reinforces that research on in vivo HAp has emerged as a significant focus in numerous countries, as illustrated in Figure 4.

The top ten authors with the highest publication counts are Xu, J. (20 documents), O’Brien, F.J. (15 documents), Liu, Q. (14 documents), Zhu, X. (14 documents), Fini, M. (12 documents), Raina, D.B. (12 documents), Zhang, X. (11 documents), Calasans-Maia, M.D. (10 documents), Lu, X. (10 documents), and Yang, X. (10 documents). This is illustrated in Figure 5.

The in vivo research on HAp, based on institutional affiliation, reveals that the leading contributors in the top 10 positions are Sichuan University (103 documents), Ministry of Education of the People’s Republic of China (88 documents), Shanghai Jiao Tong University School of Medicine (52 documents), Chinese Academy of Sciences (47 documents), Shanghai Ninth People’s Hospital, Shanghai Jiao Tong (44 documents), University School of Medicine (44 documents), Huazhong University of Science and Technology (33 documents), Southern Medical University (31 documents), Shanghai Jiao Tong University (31 documents), West China School of Medicine/West China Hospital of Sichuan University (31 documents), and Chongqing Medical University (30 documents), as shown in Figure 6.

Based on Figure 7, in the realm of in vivo research, the term “hydroxyapatite” is intricately associated with “osseointegration,” “biomechanics,” “calcium phosphate,” and “bone remodeling.” This link suggests that several in vivo investigations have focused on assessing the capacity of HAp to facilitate bone-implant integration, ^{23– 28} enhance mechanical strength, ^{29– 34} and contribute to bone remodeling and regeneration. ^{35– 39} In addition, the term “in vivo” is located in proximity to “titanium implants,” “coating materials,” and “rabbit models.” This demonstrates that in vivo experiments frequently utilize animal models, particularly rabbits, focusing on the clinical application of titanium implants covered with HAp or its derivatives.

Next, the correlation between the terms “bone substitutes,” “tibia,” and “calvarial defects” indicates that in vivo research assesses both implants and the application of HAp as a bone replacement material in diverse anatomical sites. ^{40– 44} Furthermore, from a biological standpoint, the relationship between animal cells, cell proliferation, and nanohydroxyapatite (nHAp) indicates that in vivo studies should extend beyond macroscopic evaluations to examine the cellular biological response to the material, including at the nanoscale, which is thought to enhance biocompatibility. ^{45– 47}

Figure 8 illustrates a highly centralized global collaboration network, predominantly led by the United States and China. The substantial node sizes and robust inter-country connections signify their preeminent role in the progression of HAp-based regenerative biomaterials, transitioning from conventional osteoconductive scaffolds to immunomodulatory platforms that facilitate bone regeneration, promote angiogenesis, and modulate macrophage polarization within the osteoimmune microenvironment. ^{48– 50}

In Europe (Germany, Italy, France, and the Netherlands) and East Asia (China, South Korea, and Japan), dense regional clusters were also observed, suggesting developed research ecosystems with robust intra-regional collaboration. Because contemporary HAp biomaterials depend on interdisciplinary integration, including materials science, immunology, and bioengineering to regulate immune microenvironment remodelling and enhance implant stability and long-term regenerative outcomes, such coordinated collaborations are crucial. ⁵¹

Crucially, emerging nations such as Egypt, Malaysia, Indonesia, Brazil, India, and others hold semi-peripheral positions, indicating their increasing yet still limited integration into global research networks with significant influence. This uneven allocation of collaboration capacity is crucial because new research shows that effective bone regeneration depends on precise control of immune signalling, macrophage phenotype, and ion-mediated microenvironment modulation in addition to scaffold structure. These processes necessitate sophisticated infrastructure and international cooperation. ⁵²

A small number of strongly connected innovation hubs dominate HAp research, according to the network topology overall, while new regions gradually become part of the global osteoimmunology research ecosystem. It will be crucial to improve clinical translation and expedite the development of next-generation immunomodulatory HAp biomaterials by fortifying international cooperation, especially with emerging nations.

According to journal sources that published the highest number of documents on in vivo HAp studies from 2015 to 2025, including Acta Biomaterialia, International Journal of Nanomedicine, ACS Biomaterials Science and Engineering, and Journal of Biomedical Materials Research - Part B Applied Biomaterials, International journal of biological macromolecules, Biomedical Materials (Bristol), Biomaterials, Colloids and Surfaces B: Biointerfaces, and Biomaterials Science. The top ten journals with the most substantial publications are listed in Table 1. The most frequently referenced journal was Acta Biomaterialia, with 4858 citations and an SJR of 2.007, classified in the first quartile (Q1).

HAp is a calcium phosphate with a composition analogous to bone mineral, ⁵³ rendering it extremely biocompatible and bioactive, ^{54– 56} making it an optimal choice for bone graft and bone tissue engineering applications. ^{4, 5, 57– 59} in vivo investigations were conducted to evaluate HAp’s capacity to facilitate bone remodeling and regeneration by observing new bone formation, osseointegration, and mechanical integrity. ^{60– 65} in vivo studies on HAp and its applications are summarized in Table 2.

Biologically, HAp demonstrates osteoconductivity, defined as its capacity to facilitate the migration of osteoprogenitor cells, angiogenesis, and deposition of the mineral matrix. ⁶ The application of HAp coating on titanium implants enhances bone-to-implant contact (BIC) and pullout strength in a rabbit model. ^{8, 66} The amalgamation of HAp with bioactive agents, including bone morphogenetic proteins or ion doping (e.g., copper, gadolinium, and zinc), has been documented to enhance the expression of osteogenic and angiogenic markers, expediting bone healing. ^{9, 10}

Furthermore, nHAp with interconnected porosity has demonstrated superior bone ingrowth and BV/TV ratios in critical defect models relative to traditional HAp. ^{4, 67} In addition, HAp–polymer composites (e.g., HAp-PLGA and HAp-chitosan) enhance mechanical characteristics and regulate degradation, thereby facilitating more stable bone regeneration. ¹⁰ In addition, the use of HAp coating on metallic surfaces enhances osseointegration and expedites new bone growth surrounding implants. ^{8, 68} Biphasic calcium phosphate (BCP), comprising HAp and beta-tricalcium phosphate (β-TCP), achieves an equilibrium between resorption and new bone formation, which is crucial for sustained bone remodeling. ^{12, 69} Moreover, HAp loaded with medicinal compounds, such as statins or antimicrobial compounds, exhibits a dual function of promoting osteogenesis and mitigating infection risk. ^{13, 14, 70}

Next, in vivo investigations employ mice, rabbits, or lambs based on the translational objective. ^{4, 8} Evaluation is conducted using micro-computed tomography (micro-CT) to quantify bone volume, histomorphometry to evaluate BIC and the proportion of new bone, and biomechanical testing (push-out or pull-out test) to determine functional strength. ^{6, 8, 12} Certain studies have employed immunohistochemistry to examine the expression of osteogenesis markers (RUNX2 and osteocalcin) and angiogenesis markers (VEGF). ^{10, 13}

Research reviews consistently demonstrate that nHAp and HAp-polymer composites enhance bone bridging, elevate new tissue quality, and offer superior mechanical integration compared with controls lacking HAp. ^{4, 9, 10} Ionic doping of HAp (e.g., Cu/Gd) leads to substantial enhancement of vascularization and osteoblast differentiation. ⁹ Simultaneously, tailored BCPs yield a resorption rate that aligns with the rate of new bone production, thereby diminishing the likelihood of regeneration failure. ¹²

HAp has been predominantly viewed as a passive osteoconductive material, functioning as a bone mineral analogue that promotes cell adhesion, matrix deposition, and new bone integration owing to its chemical compatibility and surface microarchitecture. ^{40, 78, 79} Diverse HAp composite scaffolds and their modifications have shown the ability to enhance osteogenesis both in vitro and in vivo by optimizing mechanical properties, degradation rates, and bioactivity. ^{56, 80, 81} Recent evidence indicates a substantial paradigm shift: HAp is not merely an osteoconductive substrate but functions as an active modulator of the immune response during the initial phase of bone healing. Furthermore, it was found that β-tricalcium phosphate particles can activate the NLRP3 inflammasome complex and enhance immune cell migration in vivo, indicating that calcium phosphate biomaterials directly interact with the innate immune pathway and influence inflammatory dynamics. ⁸²

The primary advancement is HAp’s capacity to systematically influence macrophage polarization (M1 → M2) via immunometabolic pathways. Furthermore, fluorinated HAp enhances a conducive osteo-immune milieu by inducing a metabolic transition in macrophages from glycolysis to oxidative phosphorylation, associated with the stabilization of the pro-regenerative M2 phenotype. This transition is not solely a phenotypic occurrence but offers insight into osteoblast differentiation and novel bone creation. ⁷² Moreover, HAp-coated small intestinal submucosa membranes promote periodontal regeneration via immunomodulation and the activation of the BMP-2/Smad osteogenic pathway, thereby reinforcing the notion of osteoimmunomodulation as a cohesive mechanism linking immune regulation and bone formation. ⁸³

At the biological interface level, various HAp-based scaffold designs demonstrate that successful bone regeneration is significantly influenced by the quality of interaction at the immune–bone interface, rather than merely by the material’s intrinsic osteoconductive properties. ^{84– 86} Alterations to implants designed to avert infection and enhance osseointegration ⁸⁴ and HAp-based antimicrobial delivery systems ⁸⁷ demonstrate that controlling the local inflammatory burden is crucial for facilitating the shift to the regenerative phase. Thus, the immune response is currently considered not only a secondary consequence of implantation but also a controllable factor through the chemical and surface engineering of HAp.

These studies confirm a paradigm shift from HAp as a passive osteoconductive material to an osteoimmunoengineering platform that actively modulates inflammasome signaling, promotes the M1 to M2 macrophage transition, and orchestrates immune-bone interactions to improve regeneration. This conceptual shift positions HAp within regenerative immunology, where biomaterial design prioritizes the modulation of the immune microenvironment as a crucial element in successful bone reconstruction, rather than solely concentrating on the mechanics and bioactivity of minerals.

HAp is a pivotal biomimetic material for bone replacement applications owing to its mineral composition, which closely resembles the inorganic phase of human bone, and its capacity for bioactive interactions with bone tissue. Preclinical in vivo studies are essential for evaluating the efficacy of HAp as a bone substitute, specifically regarding its capacity to promote osteogenesis, achieve osseointegration with the host bone, and restore mechanical functionality in bone defects or reconstructions. ^{4– 6, 88}

In vivo investigations elucidating the fundamental mechanisms behind HAp’s function as a bone-replacement material encompass the following: First, Osteoconduction: HAp offers a porous scaffold that promotes the migration of osteoprogenitor cells and the deposition of the mineral matrix; micro-computed tomography (μCT) and histological analyses in several studies illustrate bone ingrowth from the scaffold’s periphery towards its core. ^{5, 88} Second, surface osseointegration: HAp coating on titanium or other substrates enhances bone-to-implant contact (BIC) and mechanical fixation (pull-out/push-out) in rabbit and sheep models. ^{9, 89} Third, biological modulation by modification: Ionic doping (e.g., Sr, Cu, Gd, Zn) and loading factors (BMPs, statins, antimicrobial drugs) can augment osteoinduction and angiogenesis when the release of biomolecules/ions is regulated. ^{10, 13, 14}

In vivo investigations have evaluated diverse HAp formulations, including nHAp/3D-printed porous HAp scaffolds. Scaffolds produced through 3D printing methods or biomimetic approaches exhibit accelerated bone bridging and enhanced BV/TV in crucial defects; the primary issue lies in achieving a balance between porosity (to facilitate vascularization) and mechanical strength. ^{5, 6, 88– 90} Second, HAp–polymer composites (PLGA/PLA/collagen/chitosan) mitigate the brittleness of ceramic HAp, provide controlled degradation, and frequently demonstrate superior in vivo outcomes (vascularization and matrix quality) compared to isolated HAp. ^{91, 92} Third, HAp coatings, specifically nanostructured HAp coatings, enhance bone-implant contact (BIC) and biomechanical properties in in vivo assessments, proving especially beneficial in low-density bone scenarios. ^{9, 89} Fourth, biphasic calcium phosphate (HAp + β-TCP) modulates the resorption rate to coordinate with new bone formation, thereby enhancing long-term remodeling. ^{12, 93} Fifth, HAp serves as a drug/therapeutic carrier: HAp is optimized for the localized administration of statins, antibiotics, or anticancer drugs (e.g., 5-fluorouracil), thereby integrating osteoconductive properties with localized treatments. In vivo, it has dual therapeutic advantages without considerable systemic effects when the release is regulated. ^{13, 14} in vivo studies on HAp and its applications are shown in Table 3.

HAp has transitioned from a passive mineral carrier to a stimuli-responsive therapeutic platform that may convert problematic microenvironmental cues into programmed therapeutic responses. In pH-responsive release systems, an acidic inflammatory milieu serves as a trigger for regulated drug release, exemplified by HAp-based dual-responsive hydrogels for rheumatoid arthritis treatment, where microenvironmental circumstances govern local release kinetics. ¹⁰⁷ In the realm of infection-induced drug release, systems that respond to biofilms or reactive oxygen species (ROS) facilitate therapeutic activation solely upon an escalation of the infection burden, as evidenced by biofilm-targeting catalytic nanozymes ¹⁰⁸ and ROS-responsive antibacterial hydrogels for chronic wounds. ¹⁰⁹ This method diminishes premature release and enhances therapeutic specificity.

Moreover, in tumor microenvironments, the precise engineering of enzymes (e.g., MMP-2) facilitates tumor-specific release or activation of therapies, while concurrently altering the immunosuppressive microenvironment. ¹¹⁰ Strategies based on pH and light activation illustrate the potential of utilizing acidic conditions for selective catalytic therapy. ¹¹¹ In the osteo-immune setting, HAp alteration has demonstrated the ability to create more regenerative microenvironments by metabolically regulating immune cells, ⁷² hence validating the notion that HAp serves as a microenvironment-adaptive material. HAp in smart systems signifies a transition to precise biomaterials that not only administer medications but also modulate biological responses according to local disease dynamics.

A bibliometric analysis of worldwide in vivo HAp research shows that the field is still heavily focused on structural endpoints like implant coatings, bone remodeling, and osseointegration, with a preponderance of physicochemical optimization over immune-regenerative mechanisms. ¹ Since most in vivo studies still rely on static histological or micro-CT endpoints without longitudinal immune profiling, the mechanistic understanding of immune-bone dynamics is limited, despite growing osteoimmunology evidence that HAp can actively regulate macrophage polarization and immunometabolic reprogramming to promote bone regeneration. ^{112, 113}

Despite evidence that translational-scale models are crucial for simulating human immune responses and remodeling kinetics, bibliometric mapping further emphasizes the dominance of small-animal models with comparatively little large-animal osteoimmunology data. ^{4, 88} Furthermore, in contrast with recognized structural measurements like bone-implant contact and BV/TV, the lack of standardized immunological markers makes it impossible to compare and synthesize mechanisms across investigations. ¹¹² Finally, bibliometric trends show that AI-guided biomaterial optimization is still largely unexplored, and scaffold design still relies on empirical methods rather than predictive modeling, despite the rapid growth in material modifications such as nanostructuring, ionic doping, and composite scaffolds. ^{1, 114}

All of these experimental and bibliometric results point to a crucial translational bottleneck: long-term immune profiling, large-animal validation, standardized immune endpoints, and AI-assisted biomaterial design are necessary to move HAp from an empirically optimized osteoconductive scaffold toward a predictive osteoimmunomodulatory biomaterial.

Despite the positive results, obstacles, including diversity in animal models, discrepancies in defect size, and variations in follow-up duration, may influence inter-study comparisons. Future research should focus on optimizing scaffold porosity, integrating HAp with growth agents, and conducting long-term evaluations in large animal models before initiating human clinical trials. Consequently, in vivo investigations have substantiated the efficacy of HAp (particularly nHAp, HAp-polymer composites, and nanoscale HAp coatings) as a viable bone-replacement material, as HAp promotes bone ingrowth, osseointegration, and mechanical recovery in many animal models. Alterations (ionic doping, biphasic composition, and drug loading) may improve therapeutic efficacy and optimize resorption rates; however, clinical use necessitates long-term data from extensive models, nanoparticle safety evaluations, and standardization of in vivo study methodologies.

This bibliometric analysis confirms that HAp research remains predominantly focused in China and the US, with major trends focused on nHAp, polymer composites, and ion doping. Long-term in vivo studies using large-scale models are still needed before clinical translation.