The Effects of the Addition of Strontium on the Biological Response to Calcium Phosphate Biomaterials: A Systematic Review

Abstract

Strontium is known for enhancing bone metabolism, osteoblast proliferation, and tissue regeneration. This systematic review aimed to investigate the biological effects of strontium-doped calcium phosphate biomaterials for bone therapy. A literature search up to May 2024 across Web of Science, PubMed, and Scopus retrieved 759 entries, with 42 articles meeting the selection criteria. The studies provided data on material types, strontium incorporation and release, and in vivo and in vitro evidence. Strontium-doped calcium phosphate biomaterials were produced via chemical synthesis and deposited on various substrates, with characterization techniques confirming successful strontium incorporation. Appropriate concentrations of strontium were non-cytotoxic, stimulating cell proliferation, adhesion, and osteogenic factor production through key signaling pathways like Wnt/β-catenin, BMP-2, Runx2, and ERK. In vivo studies identified novel bone formation, angiogenesis, and inhibition of bone resorption. These findings support the safety and efficacy of strontium-doped calcium phosphates, although the optimal strontium concentration for desired effects is still undetermined. Future research should focus on optimizing strontium release kinetics and elucidating molecular mechanisms to enhance clinical applications of these biomaterials in bone tissue engineering.

1. Introduction

Nowadays, the concept of regenerative medicine is closely related to the latest research in the treatment of bone diseases and trauma. This occurs because bone pathologies are usually incapacitating, causing patients to spend many unproductive days before they reach complete recovery. Consequently, the development of treatments with reduced costs and which can accelerate recovery time is an urgent issue [1].

Due to their osteoconductive properties and bioactivity, calcium phosphate-based materials such as hydroxyapatite (HA, Ca₅(PO₄)₃(OH)), have been widely studied and used in clinics to aid in the recovery of bone lesions in treatments, such as guided bone regeneration technique [2]. One of the primary reasons for the widespread use of calcium phosphate bioceramics in bone therapy is their ability to support bone in-growth and promote the formation of new bone tissue. This is essential for the treatment of bone defects and injuries, which are often debilitating and require prolonged recovery periods. By providing a scaffold that mimics the natural bone matrix, calcium phosphate bioceramics facilitate the attachment, proliferation, and differentiation of osteoblasts, thereby accelerating the healing process and improving clinical outcomes [2]. In addition to their use in bone grafts and dental implants, calcium phosphate bioceramics are also employed in drug delivery systems, as well as in advanced fabrication techniques, such as 3D printing and nanotechnology. These technologies enable the creation of customized implants with complex geometries and controlled porosity, tailored to meet the specific needs of individual patients [2].

However, due to its high crystallinity, hydroxyapatite remains in the body for long periods after implantation, which makes complete tissue regeneration more difficult [3]. Therefore, in the search for resorbable biomaterials that can stimulate bone recovery, the partial ionic substitution of the calcium in the HA by other ions that could stimulate the production of new bone may be an essential step to achieve regenerative medicine [4]. Many ions have been experimentally incorporated into calcium phosphate ceramics, such as magnesium (Mg²⁺), zinc (Zn²⁺) and strontium (Sr²⁺) [4,5]. The presence of these components usually decreases the crystallinity of the material, making it more soluble and, since they are trace elements typically found in healthy bone tissue, there is reduced risk of adverse effects [6,7]. Among such ions, strontium has been highlighted due to the successful use of strontium ranelate in the treatment of osteoporosis [8], which indicates stimulatory biological effects during bone formation. Even though the biochemical signaling involved in this process remains unclear, it is believed that the presence of this ion can inhibit the action of osteoclasts resorption, while stimulating the production of new bone by osteoblasts [9]. Hence, the association of strontium ions with ceramic biomaterials, more than affecting physicochemical properties, may imply the direct release of biologically active material directly at the implant site.

Indeed, several studies have been conducted, either in vivo or in vitro, testing different biomaterials based on strontium-doped calcium phosphate. In this case, it is of most importance to identify the effects of strontium administration by direct interactions with resorbable biomaterials, which are most probably unrelated to the pharmacokinetic and pharmacodynamic processes already described in studies of orally administered medicaments containing Sr. In this context, the present systematic review was conducted with the objective of gathering all the latest information on the effects of strontium ion associated with ceramic materials in the bone tissue or cells, in order to determine whether its presence is, in fact, capable of stimulating bone regeneration without toxicity, as well as the data regarding the underlying mechanisms involved in strontium’s effects on guided bone regeneration.

2. Material and Methods

2.1. Database Search Strategy

This systematic review was conducted according to the protocol registered at the Open Science Framework database, available at osf.io/t9pkc, and based on the guidelines of the PRISMA Statement (Supplementary Checklist 1: PRISMA 2020 Checklist). A survey was conducted from November 2022 to June 2024, in three different databases: PubMed, Scopus and Web of Science (WoS). During the electronic search, the following Mesh (Medical Subject Headings) terms (www.nlm.nih.gov/mesh/meshhome.html (accessed on 22 August 2024)) were used to ensure the broad scope of the research: “Strontium”, “Hydroxyapatites” and “Biocompatible Materials”. The research was limited to articles published in the last ten years. Table 1 shows the search strategies used in the three databases.

Table 1. Search strategies used in the three databases.

Database	Search Key
PubMed	(“tissue engineering”[tiab] OR “Bone repair”[tiab] OR “Bone therapy”[tiab] OR “bone regeneration”[tiab] OR osteoblast*[tiab] OR “Bone tissue” [tiab] OR “bone cell*”[tiab] OR “mesenchymal stem cell*”[tiab] OR “Mesenchymal Progenitor Cell*”[tiab] OR “Bone Marrow Stromal Cells” [tiab] OR preosteoblast*[tiab] OR osteocyte*[tiab]) AND (“Strontium”[Mesh] OR SR [tiab] OR SR2+[tiab] OR strontium [tiab] OR “strontium-containing hydroxyapatite” [Supplementary Concept]) AND (“Hydroxyapatites”[Mesh] OR Hydroxyapatite*[tiab] OR Ca10(PO4)6(OH)2 [tiab] OR “tricalcium phosphate”[tiab] OR “biphasic calcium phosphate*”[tiab] OR BCP[tiab] OR beta-TCP[tiab] OR B-TCP[tiab] OR alpha-TCP[tiab] OR brushite[tiab] OR monetite[tiab] OR durapatite [tiab] OR HA[tiab] OR HAp[tiab] OR “amorphous calcium phosphate”[tiab]OR “calcium phosphate*”[tiab]) AND (“Biocompatible Materials”[Mesh] OR biomaterial*[tiab] OR “smart material*”[tiab] OR “Biomimetic Material*” OR “Biomimetic Materials”[mesh] OR “Biomimicry Material*”[tiab]) AND (biocompatib*[tiab] OR osteogen*[tiab] OR cytocompatib*[tiab] OR osteoind*[tiab] OR osteoconduc*[tiab] OR clinic*[tiab] OR surg*[tiab])
Scopus	TITLE-ABS-KEY (“tissue engineering” OR “Bone therapy” OR “bone regeneration” OR osteoblast* OR “Bone tissue” OR “bone cell*” OR “mesenchymal stem cell*” OR “Mesenchymal Progenitor Cell*” OR “Bone Marrow Stromal Cells” OR preosteoblast* OR osteocyte) AND TITLE-ABS-KEY (sr OR sr2* OR strontium OR “strontium-containing hydroxyapatite”) AND TITLE-ABS-KEY (“Biocompatible Materials” OR biomaterial* OR “smart material*” OR “Biomimetic Material*” OR “Biomimicry Material*”) AND TITLE-ABS-KEY (biocompatib* OR osteogen* OR cytocompatib* OR osteoind* OR osteoconduc* OR clinic* OR surg*) AND (LIMIT-TO (DOCTYPE, “ar”))
Web of Science	(tw:(“tissue engineering” OR “Bone repair” OR “Bone therapy” OR “bone regeneration” OR osteoblast* OR “Bone tissue” OR “bone cell*” OR “mesenchymal stem cell*” OR “Mesenchymal Progenitor Cell” OR “Bone Marrow Stromal Cells” OR preosteoblast* OR osteocyte*)) AND (tw:(sr OR sr2+ OR strontium OR “strontium-containing hydroxyapatite”)) AND (tw:(“Biocompatible Materials” OR biomaterial* OR “smart material*” OR “Biomimetic Material*” OR “Biomimicry Material*”)) AND (tw:(biocompatib* OR osteogen* OR cytocompatib* OR osteoind* OR osteoconduc* OR clinic* OR surg*)) AND (instance:”regional”) AND (type:(“article”))

2.2. Selection and Eligibility Criteria

The eligibility criteria included articles in English, Spanish, Portuguese, French and Italian, published in the last ten years, in accordance with the PECOS modified criteria, in which: P (population) = human subjects, laboratory animals or cell cultures; E (exposure) = direct or indirect exposure to calcium phosphates doped or substituted with strontium; C (comparison) = pure calcium phosphate materials; O (outcome) = altered biocompatibility, and S (setting) = In vitro or in vivo tests.

The following exclusion criteria were applied to the studies: review articles, patent applications, book chapters, theses, mixed ions, strontium not incorporated into the biomaterial, performs only physical-chemical tests, or calcium phosphate biomaterials that were mixed or integrated into complex combinations (such as polymers, bioglass, biogel, etc.).

All titles and abstracts were evaluated independently by two reviewers and selected in accordance with the eligibility criteria. Duplicates were excluded using a program that compares titles and abstracts of the papers. Articles not excluded during the abstract evaluation were thoroughly read and judged according to the eligibility criteria. Any disagreement on the eligibility of the studies was solved through discussion and consensus.

2.3. Quality Assessment of the Selected Studies and Data Extraction

Concerning the methodological quality, each one of the selected studies was evaluated using the ToxRTool (Toxicological Data Reliability Assessment Tool) as a guide for evaluations of the inherent quality of toxicity data [10]. It consists of an 18-point rating checklist, which considers the description of methodological aspects of each study, such as identification of test substance, test system, study design and results analysis. Articles that do not reach 11 points are considered unreliable, between 11–14 points are reliable with restrictions, and studies with more than 15 points are considered reliable without restrictions.

The data extraction was divided into two parts: the first corresponded to data regarding the characteristics of the biomaterial under study, including the type of material, its format, the theoretical amount of strontium added during the synthesis, the amount of strontium that is released in solution and whether it was covering titanium. The second part aimed to extract results regarding the biological response to the biomaterial. In this sense, we collected data about the cell/animal model used in the study, type of test performed, and main results obtained in comparison to experimental control. All data was used for a qualitative synthesis in the discussion section of this work.

3. Results

3.1. Database Search

The electronic search returned 1346 entries and, after the exclusion of the duplicates, a total of 883 articles were evaluated for eligibility applying the exclusion criteria. Of those, 52 studies met the established eligibility criteria and were included. The process of selection is presented in Figure 1.

Figure 1. Systematic steps according to PRISMA Statement.

3.2. Quality Assessment

Of the selected papers, all passed the quality assessment as at least reliable with restrictions and presented a good study design (Table 2). The main reasons for the lower scores in the quality assessment of the studies using the ToxRTool were due to the lack of identification of the cell origin, cell passage number, or handling methods. In vivo studies did not mention animal care practices or discussed the origin of animals but not the housing or feeding conditions. Some studies lacked either positive or negative controls. Many studies also did not report the number of replicates and provided little information on statistical tests. Furthermore, 31 articles (59.6%) presented results regarding the amount of strontium released by the biomaterial (Table 3), information that is of great importance for the adequate analysis of the results.

Table 2. Quality assessment of the in vitro and in vivo studies according to the ToXRTool.

Publication	Group I: Test Substance Identification	Group II: Test System Characterization	Group III: Study Design Description	Group IV: Study Results Documentation	Group V: Plausibility of Study Design and Data	Total
In Vitro Studies	4	3	6	3	2	18
Aina et al. [11]	4	2	5	3	2	16
Alkhraisat et al. [12]	4	2	4	3	2	15
Alkhraisat et al. [13]	4	4	6	3	2	19
Birgani et al. [14]	4	3	5	3	2	17
Boanini et al. [15]	4	2	5	3	2	16
Boanini et al. [16]	4	2	6	3	2	17
Bracci et al. [17]	4	2	5	3	2	17
Capuccini a et al. [18]	4	2	5	3	2	16
Chen et al. [19]	4	2	5	3	2	16
Chen et al. [20]	4	2	4	3	2	15
Chen et al. [21]	4	2	5	3	2	16
Chung et al. [22]	4	1	5	3	2	15
De Lima et al. [6]	4	3	6	3	2	18
Gu et al. [23]	4	3	6	3	2	18
Gu et al. [24]	4	2	6	3	2	17
Gu et al. [25]	4	2	6	3	2	17
Harrison et al. [26]	4	2	6	3	2	17
Hernández et al. [27]	4	1	5	3	2	15
Huang et al. [28]	4	3	6	3	2	18
Huang et al. [29]	4	3	6	3	2	18
Jiang et al. [30]	4	2	4	3	2	15
Jiang et al. [31]	4	3	5	3	2	17
Kuang et al. [32]	4	2	5	3	2	16
Li et al. [33]	4	2	5	3	2	16
Liang et al. [34]	4	1	3	2	2	12
Liu et al. [35]	4	3	5	3	2	17
Lourenço et al. [36]	4	2	6	3	2	17
Ma et al. [37]	4	3	6	3	2	18
Mohan et al. [38]	4	3	5	2	2	16
Nguyen et al. [39]	4	2	5	3	2	16
Ni et al. [40]	4	2	5	3	2	16
Olivier et al. [41]	4	2	5	3	2	16
Pal et al. [42]	4	2	6	3	2	17
Ramadas et al. [43]	4	2	5	2	2	15
Sartoretto et al. [44]	4	2	5	3	2	16
Stipniece et al. [45]	4	2	6	3	2	17
Sun et al. [46]	4	2	6	3	2	17
Tovani et al. [47]	4	2	5	3	2	16
Xie et al. [48]	4	2	5	3	2	16
Xie et al. [49]	4	2	3	3	2	14
Xing et al. [50]	4	2	3	3	2	14
Yang et al. [51]	4	1	4	3	2	14
Yuan et al. [51]	4	2	3	3	2	14
Zhang et al. [52]	4	3	5	3	2	17
Zhao et al. [53]	4	1	4	3	2	14
Zhao et al. [54]	4	2	5	3	2	16
Zhou et al. [55]	4	2	6	3	2	17
In Vivo Studies	4	5	6	3	2	21
Ballo et al. [56]	4	5	6	2	2	19
Chen et al. [19]	4	5	7	3	2	21
Cheng et al. [57]	4	5	7	3	2	21
Elgali et al. [58]	4	5	7	3	2	21
Gu et al. [23]	4	3	7	3	2	19
Gu et al. [24]	4	4	7	3	2	20
Ni et al. [59]	4	3	6	3	2	18
Hernández et al. [27]	4	5	6	2	2	19
Jiang et al. [31]	4	4	6	3	2	19
Li et al. [33]	4	3	6	3	2	18
Lourenço et al. [36]	4	4	7	3	2	20
Ma et al. [37]	4	4	7	3	2	20
Machado et al. [60]	4	4	7	2	2	19
Mohan et al. [38]	4	5	7	3	2	21
Ni et al. [40]	4	3	6	3	2	18
Ramadas et al. [43]	3	3	7	3	2	18
Sartoretto et al. [44]	4	5	7	3	2	21
Xie et al. [48]	4	5	7	3	2	21
Xing et al. [50]	4	5	7	3	2	21
Yan et al. [61]	4	3	7	3	2	19
Yang et al. [62]	4	3	4	3	2	16
Yuan et al. [51]	4	2	5	3	2	16
Zarins et al. [63]	4	5	7	3	2	21
Zhang et al. [64]	4	3	6	2	2	17
Zhao et al. [53]	4	3	6	2	2	17
Zhao et al. [54]	4	5	7	3	2	21

Table 3. Characteristics of the ceramic materials used in the studies.

Article	Type of the Biomaterial	Biomaterial’s Shape	Theoretical Amount of Sr in the Biomaterial (wt.%)	Amount of Sr Released from Biomaterial
Aina et al. [11]	Sr-HA	Discs	20 and 40 (at.%)	0.5–3.2 ppm (MEM culture medium) after 14 days
Alkhraisat et al. [12]	Sr Β-TCP	Cement	6.7–33 (at.%)	38–58 ppm (deionized water) after 3 days
Ballo et al. [56]	Sr-HA	Rod (titanium coating)	10.6 ug (3.55 at.%)	Not specified
Birgani et al. [14]	Sr-OCP	Scaffolds (titanium coating)	1 and 3 (at.%)	Not specified
Boanini et al. [15]	Sr-OCP	Discs (titanium coating)	10 (0.6 experimental) (at.%)	Not specified
Boanini et al. [16]	Sr-HA	Discs (titanium coating)	8.4 (experimental) (at.%)	Not specified
Bracci et al. [17]	Sr-CaP	Powder/Discs (titanium coating)	5 and 10% (3.2/5.5 experimental) (at.%)	Not specified
Capuccini a et al. [18]	Sr-HA	Discs (titanium coating)	1, 5 and 10 (0.5; 3.0 and 7.0 experimental) (at.%)	Not specified
Chen et al. [19]	Sr-CPC	Discs	1 (at.%)	1.2–1.8 ppm
Chen et al. [20]	Sr-HA	Coating on 3D printed scaffolds	Up to 8.2%	Gradually released, peaking at ~1.2 µg/mL on day 14
Chen et al. [21]	Sr-BCP	Ceramic Porous scaffolds	6.75 wt%	1.84 ppm over 6 days in media without cells
Cheng et al. [57]	Sr-CPC	Cement	0.7–2.2 (at.%)	Not specified
Chung et al. [22]	Sr-HA	Discs (titanium coating)	0, 3, 7, 15, 25, 50, 75, and 100 (at.%)	Not specified
De Lima et al. [6]	Sr-HA	Granules	1 (<0.5 experimental) (at.%)	Not detectable
Elgali et al. [58]	Sr-HA	Granules	5, 25 and 50 (~24 experimental) (at.%)	26–139 ppm (Tris-HCL) after 7 days
Gu et al. [23]	Sr-CPC	Discs	1 (at.%)	10 ppm in presence of cells/2 ppm in cell absence
Gu et al. [24]	Sr-CPC	Scaffolds	8 (at.%)	Not specified
Gu et al. [25]	Sr-HA	Coating on magnesium alloys	10%, 20%, 50%, 100%	Up to 20 µg/mL over 10 days
Harrison et al. [26]	Sr-HA	Pastes and Gels	0, 2.5, 5, 10, 50, 100 at.% Sr	Not specified
Hernández et al. [27]	Sr-HA	Cement	10 and 20% (Sr substitution)	Not specified
Huang et al. [28]	Sr-CPP	Cylinders	8% (Sr substitution)	Not indicated
Huang et al. [29]	Sr-TCP	Powder	1, 5, 10 and 15% (Sr substitution)	4.5% (PBS) after 30 days (cumulative release)
Jiang et al. [30]	Sr-HA	Discs (titanium coating)	10%	~1.5 ppm (PBS) after 9 days
Jiang et al. [31]	Sr-HA	Disc	2.5%, 5%, 10%, 20%	0.2–1.0 µg/mL over 4 days
Kuang et al. [32]	Sr-CPC	Cement	5, 10 and 20 (at.%)	Not specified
Li et al. [33]	Sr-HA	Scaffolds (titanium coating)	10, 40 and 100% (9.14, 37.80 and 100 experimental)	Not specified
Liang et al. [34]	Sr-Brushite	Rod (titanium coating)	5, 10 and 20 (at.%)	Not indicated
Liu et al. [35]	Sr-CPP	Cylinders	1, 2, 5, 8 and 10 (at.%)	Not indicated
Lourenço et al. [36]	Sr-HA	Powders	5%, 10%, 15%, 20%	Not specified
Ma et al. [37]	Sr-HA	Coating on PET artificial ligaments	2, 4, 6, 8, 10 mol%	6.33, 7.48, 11.23, 14.96, 20.26 mg/mL (PBS) after 30 days
Machado et al. [60]	Sr-HA	Spheres	0.71 (at.%)	Not specified
Mohan et al. [38]	Sr-HA	Discs	50 (at.%)	~8 ppm (SBF) and 20 ppm (PBS) after 28 days
Ni et al. [40]	Sr-HA	Powder	1, 5 and 10% (Sr substitution)	5.05, 12.7 and 19.46 ppm in media after 24 h.
Olivier et al. [41]	Sr-HA	Coatings on Activated Carbon Fiber Cloth	5% and 10%	Not specified
Pal et al. [42]	Sr-HA	Powders and Pellets	10%, 30%, 50%, 70% of Ca replaced by Sr	Not specified
Ramadas et al. [43]	Sr-HA	Porous Scaffold	2.8 wt%	Not specified
Sartoretto et al. [44]	Sr-CHA	Microspheres	5 (at.%)	~4 ppm (culture medium) after 24 h
Stipniece et al. [45]	Sr-nHAp	Powder	1, 3 and 10 wt%	up to 1 mg/mL
Sun et al. [46]	Sr-α-TCP cement	Disc	8.3 and 16.7%	Not specified
Tovani et al. [47]	Sr-CaP	Nanotubes	10% and 50 at.%	2–18 mg/L
Xie et al. [48]	Sr-CPC	Scaffolds	8 (at.%)	~0.020 ppm (SBF) after 4 weeks
Xie et al. [49]	Sr-CaP	Scaffold	4.2%	Not specified
Xing et al. [50]	Sr-CaP	Coating on titanium alloy	2 wt%	Not specified
Yan et al. [61]	Sr-HA	Rod (titanium coating)	5, 10 and 20 (7.6, 13 and 22.7 experimental)	Not specified
Yang et al. [62]	Sr-HA	Disks	(9.1, 91 and 98.6 experimental) (at.%)	Not specified
Yuan et al. [51]	Sr-HA	Injectable Gel	15 mol%	Not specified
Zarins et al. [63]	Sr-HAP/TCP	Granules	5 wt%	Not specified
Zhang et al. [52]	Sr-HA	Discs	10, 40 and 100 (8.73, 37.95 and 100 experimental) (at.%)	14, 35 and 50 ppm (DMEM) after 24 h.
Zhang et al. [64]	Sr-HA	Cylinders (titanium coating)	2.5 (at.%)	~0.53 ppm (NaCl 0.9%) after 24 days
Zhao et al. [53]	Sr-ABS	Scaffolds	~2–5 experimental (at.%)	~5 ppm (deionized water) after 5 days
Zhao et al. [54]	Sr-HA	Whisker-like Scaffolds	10%	1.5–2.0 ppm (in vitro, over 7 days)
Zhou et al. [55]	Sr-HA	Scaffolds (titanium coating)	16.5 (at.%)	Not specified

Sr-OCP = octacalcium phosphate; Sr-CaP = strontium calcium phosphate; Sr-CPC = strontium calcium cement; Sr Β-TCP = strontium B-tricalcium phosphate; Sr-TCP = strontium tricalcium phosphate; Sr-CPP = strontium calcium polyphosphate particles; Sr-ABS = Allograft Bone Scaffolds.

The main types and presentations of the biomaterials, as well as their theoretical concentrations of strontium added or incorporated, are presented in Table 3, ranging from powders to scaffolds for bioengineering purposes, and presenting from 0.5 to 100% Sr substitution in the calcium site. Most studies presented at least an FTIR (Fourier-transformed infrared spectroscopy) analysis, demonstrating the chemical composition of each studied material, which guarantees its purity, as well as its ceramic nature.

The biomaterials were primarily in the form of discs, rods, scaffolds, powders, and coatings on various substrates, including titanium and magnesium alloys. These materials were characterized for their strontium incorporation and release properties, which are crucial for their performance in bone regeneration applications. The amount of strontium released from these biomaterials was measured in various ways, indicating the importance of controlled release for achieving the desired biological effects without toxicity.

Table 4 shows the main in vitro tests used to assess cytocompatibility and the effects of the addition of strontium ions to ceramic materials in different cell types, mainly of osteoblastic lineage. It is possible to note that in addition to cell viability tests such as MTT and LDH, the most evaluated parameter was alkaline phosphatase (ALP). This protein can be analyzed by dosing its presence or by demonstrating its activity. The preference for the evaluation of ALP activity was notorious since only one article evaluated its presence with a specific dye [35].

Table 4. Parameters evaluated on the in vitro studies.

Article	Cell Type	Test Performed	Effects
Aina et al. [11]	Human osteoblast	ALP, LDH and H3-thymidine incorporation	Increased osteoblast differentiation
Alkhraisat et al. [12]	Human osteoblasts (hFOB1.19)	Electronic cell counting and WST1	Similar to the control
Birgani et al. [14]	hMSCs	ALP, cell morphology	Increased ALP activity; smaller cell area
Boanini et al. [15]	Osteoblast-like (MG-63)	WST1, ALP, Type 1 collagen, OSC and Cell morphology	Increased osteoblast differentiation
Boanini et al. [16]	Osteoblast and osteoclast	WST1, ALP, Type 1 collagen, OPG, TRAP, RANKL and Cell morphology	Increased osteoblast differentiation
Bracci et al. [17]	Osteoblast-like (MG-63)	WST1, LDH, ALP, Type 1 collagen, OSC and cell morphology	Increased osteoblast differentiation
Capuccini a et al. [18]	Osteoblast-like (MG63)/Osteoclasts (human)	WST1, ALP, Type 1 collagen, OSC, OPG OSC, TRAP and cell morphology	Decreased formation of osteoclasts/increased osteoblast differentiation
Chen et al. [19]	Human endothelial cells (ECV304)	SEM, MTT, migration activity	Increased migration and proliferation
Chen et al. [20]	MC3T3-E1 osteoblasts	Cell attachment, proliferation (CCK-8 assay), differentiation (ALP activity, RT-PCR for Col I, Runx-2, OPN, and Osterix)	Enhanced cell attachment, proliferation, increased ALP activity, and upregulated expression of osteogenic markers (Col I, Runx-2, OPN, and Osterix)
Chen et al. [21]	RAW 264.7 macrophages, mMSCs	Cell proliferation (AlamarBlue assay), cell morphology (CLSM, SEM), gene expression (RT-PCR), protein expression (ELISA, Western blot)	Enhanced osteogenic differentiation, inhibited osteoclastic differentiation, upregulated osteogenic gene expression, downregulated osteoclast-specific protein activity (TRAP, CAII)
Chung et al. [22]	MC3T3-E1 osteoblast/RAW264.7 osteoclast	MTT and cell morphology.	Decreased formation of osteoclasts/increased osteoblast differentiation
De Lima et al. [6]	Balb/c 3T3 fibroblasts/Primary human osteoblasts	XTT, NR, CVDE, apoptosis induction and cell morphology and adhesion.	Increased osteoblast differentiation
Gu et al. [23]	Murine macrophage (RAW264.7)/rabbit osteoclasts	MTT, TRAP and SEM	Increased macrophage-mediated degradation/inhibited osteoclasts activity
Gu et al. [24]	Endothelial cells/primary human osteoblasts	MTT, TLS	Increased proliferation and TLS formation
Gu et al. [25]	MC3T3-E1 osteoblasts	Cell proliferation (CCK-8 assay), ALP activity, fluorescent staining	Enhanced proliferation, higher ALP activity, improved cell morphology with increasing Sr content
Harrison et al. [26]	MG63 human osteoblast-like cells	Direct biocompatibility (PrestoBlue® assay, Thermo Fisher Scientific, Waltham, MA, USA), indirect biocompatibility (PrestoBlue® assay)	High viability for indirect biocompatibility; direct biocompatibility affected by paste/gel disaggregation, highest viability observed with 0 and 100 at.% SrHA
Hernández et al. [27]	Human fibroblasts	MTT, Alamar blue, LDH, and SEM	Increased cytotoxicity
Huang et al. [28]	hMSCs	Cell counting and WST8	Increased proliferation
Huang et al. [29]	Osteoblasts (ROS17/2.8)/macrophages (RAW264.7)	OPG and RANKL	More secretion of OPG after 48 h and less secretion of RANKL after 24 h
Jiang et al. [30]	MC3T3-E1 and rat’s BMSC	Flow cytometry, ALP, OCN, alizarin red	Increased differentiation and proliferation
Jiang et al. [31]	Bone marrow stromal cells (BMSCs)	Cell adhesion, proliferation (MTT), ALP activity, gene expression (qRT-PCR), protein expression (Western blot)	Enhanced adhesion, proliferation, ALP activity; increased expression of COL1, BSP, BMP-2, OPN, VEGF, ANG-1
Kuang et al. [32]	Osteoblast-like (MG63)	WST1, ALP, and SEM	Higher proliferation rate and ALP activity of MG-63
Li et al. [33]	MC3T3-E1 osteoblasts	Cell adhesion, proliferation (MTT), ALP activity, gene expression (Runx2), protein expression (OPN, OCN)	Enhanced adhesion and proliferation, increased ALP activity, higher Runx2 gene expression, and elevated OPN and OCN protein levels
Liang et al. [34]	Osteoblast (MC3T3-E1)	MTT	Increased proliferation in the 5 and 10% of Sr
Liu et al. [35]	Osteoblast (ROS17/2.8)	ALP, MTT and SEM	Increased proliferation
Lourenço et al. [36]	Human adipose-derived stem cells	Cell viability, differentiation (ALP activity, mineralization)	Sr-modified scaffolds promoted cell viability and differentiation, with increased mineral deposition compared to control
Ma et al. [37]	Rat Bone Marrow Stem Cells (rBMSCs)	ALP activity, ARS staining, real-time PCR (RT-PCR)	Increased ALP activity and mineralization in 2SrHA-PET group.
Mohan et al. [38]	Adipose-MSCs	Mineralization, ALP, SEM, CLS and Micro-CT	Increased differentiation and proliferation
Nguyen et al. (2018)	Mouse osteoblast-like cells (MC3T3-E1)	Cell attachment, cell proliferation (CCK-8), cell morphology (CLSM)	Enhanced cell attachment, better proliferation on ASH55 group with 20 cycles, improved cell morphology
Ni et al. [40]	Fibroblasts (L-929)	Fluorescense microscopy	non-cytotoxic
Olivier et al. [41]	Human primary osteoblasts	Cell viability and proliferation (calcein-AM and ethidium-homodimer-1 assay)	Sr-doped coatings (5% and 10%) showed significant improvement in cell proliferation compared to non-doped coatings
Pal et al. [42]	Mouse osteoblast (MC3T3-E1)	MTT assay for cytotoxicity, cell proliferation	Non-cytotoxic, cell proliferation decreases with more than 50% Sr substitution
Ramadas et al. [43]	MG-63 osteoblasts	Cytotoxicity (MTT assay), cell viability	Cell viability significantly reduced with increased concentration of scaffolds (93–45% for 10 and 1000 μg/mL, respectively)
Sartoretto et al. [44]	Pre-osteoblastic MC3T3-E1 cells	MTS, Runx2, Osterix, ALP, Collagen 1a1	Higher than control (MTS assay); significant upregulation in osteogenic medium treated groups (qPCR analysis)
Stipniece et al. [45]	MG-63 osteoblasts	ALP activity, gene expression (COL1, OCN, OPN)	Increased ALP activity, expression of collagen I and osteocalcin indicating boosted bone formation
Sun et al. [46]	MC3T3-E1 pre-osteoblasts	Cell proliferation (MTT), cytotoxicity test	Enhanced proliferation, lower cytotoxicity at lower Sr concentrations
Tovani et al. [47]	Pre-osteoblastic MC3T3-E1 cells; bone marrow macrophages (BMMs)	Cell viability (MTT assay), ALP activity (ELISA), Osteocalcin expression (PCR; Osteoclastic differentiation (TRAP activity))	Enhanced cell viability and differentiation, increased ALP activity and osteocalcin expression; dose dependent activation of osteoclasts
Xie et al. [48]	Osteoblast-like (ROS17/2.8)	MTT	Similar to the control
Xie et al. [49]	MC3T3-E1 osteoblasts	Cell proliferation assay, ALP activity assay, Alizarin red staining	Increased proliferation, higher ALP activity, and greater mineralization under high calcium conditions
Xing et al. [50]	Rabbit bone marrow stromal cells (rBMSCs)	Cell adhesion assay, cell proliferation assay, ALP activity assay, Alizarin red staining	Enhanced cell adhesion, increased proliferation, higher ALP activity, and greater mineralization in Sr-CaP-p group
Yang et al. [62]	Osteoblast (MC3T3-E1)/primary rabbit osteoclasts	Alamar Blue, ALP and resorption pits	Decreased resorption pits and ALP expression, similar to the control
Yuan et al. [51]	Mouse Raw 264.7 macrophages, MC3T3-E1 osteoblasts	MTT assay, gene expression (IL-1β, IL-6, TNF-α), cytokine secretion (ELISA for IL-1β, IL-6, TNF-α, RANTES, MCP-1, MIP-1α), real-time PCR (OPG, ALP, OCN, COL-I, c-fos)	Decreased macrophage proliferation, down-regulated gene expression and cytokine secretion of IL-1β, IL-6, TNF-α, increased osteoblast viability, and osteogenic gene expression
Zhang et al. [52]	Osteoblast-like (MG63)	MTT and ALP	The 10% Sr-HÁ promoted proliferation while higher concentrations decreased it
Zhao et al. [53]	Fibroblasts (L929)	MTT	Similar to the control
Zhao et al. [54]	Pre-osteoblastic MC3T3-E1 cells	ALP staining, real-time PCR	Increased osteoblast differentiation
Zhou et al. [55]	Rat MSCs	CCK-8, ALP, OCN, OPN and Type 1 collagen	Interrod spacing larger than 137 nm inhibited in vitro mesenchymal stem cell functions

OSC = osteocalcin; SEM = scanning electron microscopy; OPG = osteoprotegerin; TLS = tube-like structure; CLS = confocal laser scanning.

Most in vitro tests performed at least two assays, covering both viability and cell differentiation analysis. The exception was the articles by Zhao et al. [65], Liang et al. [34], and Xie et al. [48], which performed only the MTT test (Table 3). However, all these studies performed in vivo tests, which presented analyses of bone regeneration. Xie et al. [48] also presented molecular tests that could replace the in vitro tests of cell differentiation.

The results from in vivo studies among the selected papers were summarized in Table 5. Most of the authors performed histological studies to investigate the structure of the tissue surrounding the grafted areas. However, new X-ray techniques were also widely used to evaluate the implant area. This strategy is favorable since it can be used with living animals, which allows the analysis of the entire lesion recovery process. Table 4 also compiles tests that covered signaling pathways that could aid in the understanding of the effect of strontium on the bone tissue when associated with ceramic materials. Some authors have sought to elucidate possible effects on the expression of genes related to bone differentiation, among them osteocalcin (OSC), osteopontin (OPN), and bone sialoprotein (BSP). Other studies focused on the inflammatory pathway by dosing cytokines such as TNF-α or on revascularization by dosing VEGF.

Table 5. Parameters evaluated in the in vivo studies.

Article	Animal Type	Test Performed	Effects
Ballo et al. [56]	Sprague-Dawley rats	Histology, histomorphometry, and SEM	Increased new bone formation
Chen et al. [21]	Male Balb/c mice	Intramuscular implantation of Sr-BCP scaffolds; histological analysis, TRAP staining, IHC staining for CTSK	Enhanced ectopic osteogenesis and reduced osteoclastogenesis compared to BCP scaffolds
Chen et al. [21]	Sprague-Dawley rats	3D fused PET-CT image	Similar to the control
Elgali et al. [58]	Rats	Histology, histomorphometry, and immunohistochemistry	Increased bone area
Gu et al. [23]	New Zealand white rabbits	VEGF histological marking	Increased angiogenesis
Gu et al. [24]	New Zealand white rabbits	Histology, X-ray	Accelerated bone repair
Hernández et al. [27]	Wistar rats	Histology	Similar to the control
Jiang et al. [30]	Rat calvarial defect model	Micro-CT, histological analysis	Enhanced bone and blood vessel regeneration, highest in 10% Sr-doped mnHAp group
Li et al. [33]	New Zealand rabbits	3D-CT analysis, histological evaluation	Significant bone formation and faster degradability in Sr10-TBC group, new bone area ratio higher in Sr10-TBC group compared to TBC
Liang et al. [34]	Sprague-Dawley rats	X-ray, Micro-CT, removal torque	Accelerated bone repair in the 10% concentration and increased removal torque (5 and 10%).
Lourenço et al. [36]	Rat Critical-Sized Defect Model	Bone regeneration	Promoted bone regeneration, modulated immune response towards M2 macrophage phenotype, induced collagen formation around implants
Machado et al. [60]	Santa Ines sheep	X-ray microfluorescence, SEM histology and histomorphometry	Similar to the control
Mohan et al. [38]	New Zealand rabbits	X-ray, Micro-CT, histology, and histomorphometry	Increased bone formation
Ramadas et al. [43]	Rabbit tibia bone defect model	Histological analysis	Significant bone mineralization process, scaffold replaced with fibrous tissue, including trabecular and spongy bone tissues, vascular tissue
Sartoretto et al. [44]	WT and ASC KO mice	Subcutaneous and tibia implantation; histomorphometry	Bone formation (% new bone) in Sr-containing groups higher in WT than ASC KO, CHA > HA for WT; degradation of biomaterial in vivo higher in CHA, similar between SrCHA and HÁ
Xie et al. [48]	New Zealand rabbits	X-ray microradiography	Increased new bone formation after eight weeks
Xie et al. [49]	Rabbits	Histological analysis, Micro-CT	Enhanced bone formation and osseointegration under high calcium conditions
Xing et al. [50]	Rabbits	Histological analysis, Micro-CT	Improved bone formation and osseointegration in Sr-CaP-p group compared to control
Yan et al. [61]	New Zealand white rabbits	Micro-CT, histology and pull-out test.	The 20% SrHA promoted better bone–implant integration and new bone formation.
Yuan et al. [51]	Ovariectomized rat femoral defect model	Surgical procedure, micro-computed tomography (µ-CT), histological assessment, immunohistochemical staining for IL-6	Enhanced new bone formation with 15SrHA/G3-K PS group, lower gene expression of IL-1β, TNF-α, and IL-6, increased IL-6 expression in HA/G3-K PS group
Zarins et al. [63]	Rabbits	Histological and immunohistochemical analysis	Enhanced bone regeneration, increased expression of OC, OPG, NFkB 105, BMP 2/4, and Col-1α in the peri-implant zone of Sr-enriched HAP/TCP group compared to non-operated leg and sham surgery groups
Zhang et al. [52]	Sprague-Dawley rats	histomorphometry	Increased bone-to-implant contact and new bone apposition
Zhao et al. [53]	New Zealand white rabbits	Compressive strength and confocal microscopy	Similar to the control
Zhao et al. [54]	Osteoporotic rat model	Histological analysis, μCT analysis	Enhanced bone regeneration in osteoporotic defects, substantial vascular-like structures, increased new bone formation
Zhou et al. [55]	New Zealand rabbits	Histology	Interrod spacing larger than 137 nm inhibited in vivo osseointegration

4. Discussion

4.1. Functionalization with Strontium

When a biodegradable biomaterial is functionalized with a particular ion such as strontium, it is expected that its release in the intercellular medium induces some biological response, activating or deactivating specific cellular pathways. It is known that as in any therapeutic process, the levels of strontium ion release should be within a specific range so that its effect on the tissue is beneficial without presenting toxicity to the organism [66].

The diversity in the shapes and types of strontium-doped calcium phosphate biomaterials, as summarized in Table 3, plays a significant role in their efficacy for bone therapy and material testing. Discs and rods, for instance, provide a standardized geometry that is ideal for in vitro testing of cellular responses, such as proliferation, differentiation, and cytotoxicity. Studies using discs, like those by Aina et al. [11] and Boanini et al. [16], demonstrated enhanced osteoblast differentiation and biocompatibility, which are critical for preliminary material evaluation.

Scaffolds, on the other hand, mimic the three-dimensional structure of bone more closely and are thus more relevant for in vivo applications. For example, the porous scaffolds used by Chen et al. [21] and Gu et al. [24] showed significant improvements in new bone formation and angiogenesis, highlighting their potential for effective bone regeneration therapies. The use of scaffolds allows for better integration with the host tissue, facilitating cell infiltration and vascularization, which are essential for successful bone healing. Additionally, scaffolds can be fabricated using advanced techniques like 3D printing, employing nanomaterials to create complex anatomical features for bone therapy [67].

Powdered materials, as utilized by Boanini et al. [15] and Alkhraisat et al. [12], offer versatility in being mixed with various carriers or used as coatings. This flexibility can be advantageous in both research and clinical settings, allowing for customized application methods tailored to specific needs. The release kinetics of strontium from these powdered materials also provide valuable insights into the material’s behavior in biological environments, as nanostructured materials may release biologically active particles after their disintegration during the material absorption, as seen in studies where controlled release profiles were crucial for maintaining non-toxic yet therapeutic levels of strontium [12,15]. Often, nanostructured materials and nanoparticles present biological effects of relevance to biocompatibility [68].

Coatings on substrates like titanium and magnesium alloys, examined by studies such as those by Gu et al. [25] and Liang et al. [34], are particularly relevant for orthopedic and dental implants. These coatings enhance the surface properties of the implants, promoting better osseointegration and reducing the risk of implant failure. The ability to control the strontium content and its release from these coatings ensures that the therapeutic benefits are maximized while minimizing potential adverse effects.

In this review, it was possible to identify that very different amounts of strontium were incorporated into the biomaterials, varying from 0.5 to 100% substitution at the calcium site. However, the biological effect found is not directly related to the theoretical amount of strontium added since similar substitution amounts presented opposite effects. For example, in a comparison between Kuang [32] and Liang [34] that have the same Sr substitution amount (5, 10, and 20%), it can be seen that the former does not find statistical difference during an in vitro analysis between the different Sr concentrations at the biomaterial, whereas in the latter there is a significant decrease in the number of osteoblastic cells in contact with the conditioned medium with the material with 20% strontium substitution.

These differences may be due to characteristics of the materials tested since they have different dissolution rates. The beneficial stimulus effect for osteogenesis of the supplementation of the culture medium with strontium is dose-dependent [69]. During in vitro experiments, a Sr supplementation of 2–5 ppm in the culture medium was able to stimulate osteoblastic cells to form mineralization and calcification nodules. However, a Sr concentration of 20–100 ppm inhibits the calcification [69]. Thus, a more soluble biomaterial releases a higher concentration of strontium in the environment, which combined with the strontium dose-dependent effect can determine the cellular behavior.

In addition to that, bone tissue presents a particular type of cell, the osteoclasts, with the function of resorbing the mineral part of the bone. Therefore, it is expected that in their presence, the release of strontium ions is much higher than in their absence. However, a single article investigated in vitro the amount of Sr released in the presence of these cells and, in fact, the amount of strontium in the culture medium rose from 2 mg/dL in the absence of cells to 10 mg/dL when the biomaterial was in contact with mature osteoclasts [24]. Elgali et al. [58] identified the presence of osteoclasts in direct contact with material granules. It was hypothesized that ceramic materials that have a high percentage of substitution by strontium ions can generate a peak of Sr during the active reabsorption by osteoclasts, which may or may not be toxic in the microenvironment, even though the amount of strontium released into the environment was not evaluated.

According to Dahl et al. [9], the incorporation and distribution of strontium in the skeleton depend on factors such as gender, skeletal size, the dose administered, and duration of treatment with strontium ranelate. None of these factors was addressed in the studies about the effect of strontium associated with bioceramics. Therefore, the study of the amount of strontium that must be added to the biomaterial is essential for medical application so that it releases in the biological medium, and the presence of osteoclasts, a safe and also effective amount of strontium.

The biological responses to the investigated biomaterials may be dependent on the level of Sr incorporation. Nguyen et al. [39] observed that titanium biomedical devices coated with strontium-doped calcium phosphate ceramics supported desirable bone regeneration by enhancing cell adhesion and proliferation. The study indicated that sufficient strontium doping concentrations in the coatings could significantly improve cellular attachment and proliferation, making these coatings promising for bone regeneration applications. Gu et al. [25] demonstrated that strontium-substituted hydroxyapatite coatings on magnesium alloys significantly controlled the degradation rate and enhanced the osteoblast response in vitro. The study indicated that the Sr content in the coatings could be adjusted to optimize the degradation behavior and cytocompatibility of Mg alloys for biomedical applications. Li et al. [33] found that strontium-doped hydroxyapatite coatings on titanium implants significantly enhanced the osteogenic differentiation of human bone marrow mesenchymal stem cells and improved osseointegration in rabbit models. That study emphasized the importance of optimizing strontium concentrations in hydroxyapatite coatings to maximize their osteogenic potential. Lourenço et al. [36] reported that strontium-substituted hydroxyapatite coatings on titanium implants significantly enhanced osseointegration and bone-implant contact in rabbit models. The study concluded that the optimal concentration of strontium in hydroxyapatite coatings was crucial for maximizing bone regeneration while minimizing potential adverse effects.

The incorporation of Sr may impact the structural properties of calcium phosphate biomaterials. Pal et al. [42] revealed that the synthesis of strontium-doped hydroxyapatite from Mercenaria clam shells enhanced mechanical properties such as microhardness and fracture toughness while maintaining excellent biocompatibility. The Ca/Sr ratio may also be relevant both to the structure and biological performance of the biomaterial. Xie et al. [49] reported that strontium inhibits bone regeneration under low calcium concentrations but promotes it under high calcium concentrations, suggesting that the calcium-strontium balance is relevant to optimize bone healing outcomes.

The reviewed studies indicate that the incorporation of strontium into calcium phosphate-based biomaterials at different strontium concentrations influence cytotoxicity, osteoblast differentiation, proliferation, osteoclast inhibition, and new bone formation. The biological effects may be dose-dependent, as demonstrated by Zhao et al. [54], who showed that strontium-doped scaffolds not only enhanced new bone formation in osteoporotic rat models but also increased vascular-like structures, indicating a dose-dependent osteogenic and angiogenic response to strontium release from the biomaterial. Olivier et al. [41] found that strontium-doped bioceramics enhanced osteogenic differentiation and mineralization in mesenchymal stem cells, supporting their potential for use in bone tissue engineering, suggesting dose-dependent effects of strontium on cellular responses and emphasizing the importance of optimizing strontium concentrations in biomaterials for therapeutic applications [41].

Figure 2 highlights the concentration ranges of strontium ion incorporation and their respective biological impacts, facilitating a better understanding of the dose-dependent nature of strontium in these biomaterials. At lower concentrations (0.5% to 2.5%), the biomaterials exhibit non-cytotoxic properties, making them safe for cellular applications. In this range, there is a significant enhancement in osteoblast differentiation and proliferation, indicating that low doses of strontium can effectively promote bone cell activity and growth. Additionally, new bone formation is highly stimulated, suggesting that these concentrations are optimal for bone regeneration applications. This is consistent with studies such as those by Aina et al. [11] and Boanini et al. [16], which reported enhanced osteoblast activity and biocompatibility at these concentrations.

Figure 2. Expected biological effects of different ranges of strontium ion incorporation into calcium phosphates, according to the evidence from literature.

In the moderate concentration range (2.5% to 5%), the biomaterials continue to exhibit non-cytotoxic effects. Osteoblast differentiation remains high, and proliferation is moderate to high, supporting the earlier findings that strontium enhances cellular activities conducive to bone regeneration. Moderate osteoclast inhibition is observed, which helps in balancing bone formation and resorption. The new bone formation remains high, making this range suitable for therapeutic applications that require robust bone regeneration, as supported by studies from Chen et al. [21] and Gu et al. [25].

As the concentration increases to the intermediate range (5% to 10%), there is a noticeable shift in the biological effects. Low levels of cytotoxicity begin to appear, indicating that higher strontium concentrations may pose some risks to cell viability. Osteoblast differentiation and proliferation are moderate, while osteoclast inhibition remains effective. New bone formation continues to be high, though it is crucial to monitor cytotoxic effects at these concentrations. This range is a critical balance point where the benefits of bone regeneration need to be weighed against the potential cytotoxic risks. At high concentrations (10% to 20%), the cytotoxic effects become more pronounced, with moderate cytotoxicity levels observed. Osteoblast differentiation and proliferation decrease significantly, indicating that high doses of strontium may inhibit these crucial processes. Osteoclast inhibition is low, and new bone formation is moderate, suggesting that while some regenerative effects are still present, the overall efficacy is reduced due to the adverse effects on cell viability and activity. These findings align with the studies by Liang et al. [34], which reported reduced cellular activities at higher strontium concentrations.

At very high concentrations (20% and above), the biomaterials exhibit high cytotoxicity, severely limiting their applicability in bone regeneration. Osteoblast differentiation and proliferation are minimal, and osteoclast inhibition is negligible. New bone formation is also minimal, indicating that such high doses are detrimental to both cell viability and the overall bone regeneration process. This concentration range should be avoided in therapeutic applications due to the significant cytotoxic effects and reduced regenerative potential. These findings underscore the importance of selecting appropriate strontium concentrations to maximize therapeutic benefits while minimizing potential adverse effects. Further research should focus on refining these concentration ranges and exploring the underlying mechanisms to optimize the use of strontium-doped calcium phosphate biomaterials in clinical applications.

4.2. Strontium Release

The correlation between the functionalization of the ceramic with the strontium ion and the expected effects of osteoinduction may lie in its ability to release the ion in the intercellular medium [70]. The release of strontium ions (Sr) from calcium phosphate-based biomaterials is a critical factor influencing their biocompatibility and therapeutic effectiveness. In our systematic review, we observed that 29 out of 52 included studies provided detailed records on the Sr release from the biomaterials. This section discusses the release profiles of these materials and their implications for biological responses, particularly in bone regeneration.

Strontium release profiles varied significantly across different studies, reflecting the diverse nature of the biomaterials and their preparation methods. For instance, Jiang et al. [31] reported that Sr-HA discs released between 0.2 to 1.0 ppm over four days, while Ni et al. [40] observed releases ranging from 5.05 to 19.46 ppm in media after 24 h. This variability indicates that the structural properties and composition of the biomaterials play a crucial role in determining the release kinetics of Sr ions.

The controlled release of Sr is essential for achieving the desired biological effects without causing toxicity. Studies such as those by Ma et al. [37] highlighted that Sr-HA coatings on PET artificial ligaments released 6330 to 20,260 ppm over 30 days, which was beneficial for sustained therapeutic outcomes. Conversely, high concentrations of Sr release, such as the 38–58 ppm observed by Alkhraisat et al. [12] in Sr-β-TCP cement, could potentially lead to cytotoxic effects if not carefully regulated.

The beneficial effects of Sr release on bone formation and cellular responses are dose-dependent. For example, controlled releases of Sr in the range of 2–5 ppm have been shown to stimulate osteoblastic activity and enhance mineralization, as demonstrated by multiple studies. On the other hand, excessive release, such as 20–100 ppm, has been associated with inhibitory effects on calcification and potential cytotoxicity.

Interestingly, only a few studies, such as Gu et al. [23], investigated the dynamic release of Sr in the presence of osteoclasts, showing an increase from 200 ppm to 1000 ppm when in contact with these bone-resorbing cells. These findings underscore the importance of considering the biological environment in which the biomaterials will be used, as the presence of specific cell types can significantly alter the release dynamics and subsequent biological effects. Therefore, the release profile of strontium from calcium phosphate-based biomaterials is a critical parameter that needs careful optimization. Ensuring a controlled and sustained release of Sr can maximize the therapeutic benefits for bone regeneration while minimizing potential adverse effects.

4.3. In Vitro Studies

Several studies observed that strontium-doped biomaterials significantly improved osteogenic differentiation and biocompatibility. For instance, Xing et al. [50] found that air-plasma-treated titanium alloy coatings with strontium-doped phosphate significantly improved osteogenic differentiation and biocompatibility. Similarly, Chen et al. [20] showed that strontium-doped mesoporous bioactive glass scaffolds significantly promoted osteoblast differentiation and angiogenesis, suggesting their potential for enhancing bone regeneration through dual osteogenic and angiogenic effects. Chen et al. [21] observed that strontium-substituted hydroxyapatite coatings on polyetheretherketone (PEEK) significantly enhanced the proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs), highlighting their potential for improving the biological performance of orthopedic implants. In addition, Lourenço et al. [36] demonstrated that strontium-substituted hydroxyapatite coatings significantly enhanced osteogenic differentiation and mineralization in mesenchymal stem cells, emphasizing the need for precise control over strontium concentration to optimize cellular responses. Ma et al. [37] confirmed that strontium-substituted hydroxyapatite coatings significantly improved the proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs), highlighting the potential of strontium-doped biomaterials for enhancing bone regeneration in vitro.

Other studies confirmed interesting effects in different types of Sr-doped calcium phosphates. Pal et al. [42] found that the Sr-doped hydroxyapatite derived from Mercenaria clam shells not only improved mechanical properties but also enhanced osteoblast proliferation and differentiation, demonstrating excellent in vitro biocompatibility and potential for bone tissue engineering. Nguyen et al. [39] observed that titanium substrates coated with strontium-doped calcium phosphate ceramics exhibited enhanced cell adhesion and proliferation, emphasizing the importance of optimizing the strontium doping concentration. Jiang et al. [31] showed that strontium-doped hydroxyapatite bioceramics with micro-nano-hybrid surfaces significantly promoted the proliferation, differentiation, and angiogenic activity of bone marrow stromal cells (BMSCs), highlighting the synergistic effects of surface modification and strontium doping. Li et al. [33] demonstrated that strontium-doped hydroxyapatite coatings on titanium substrates significantly enhanced the proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs), suggesting their potential for improving bone regeneration in vitro. Harrison et al. [26] observed that strontium-doped bioceramics significantly enhanced the expression of osteogenic markers and promoted bone regeneration in vitro, indicating their potential for improving the biological performance of biomaterials for bone repair applications.

Sun et al. [46] developed a novel fast-setting strontium-containing hydroxyapatite bone cement with a simple binary powder system. This new Sr-CPC displayed excellent compressive strength and favorable cytocompatibility, highlighting its potential for clinical applications in bone tissue repair. Stipniece et al. [45] further demonstrated that Sr-functionalized hydroxyapatite nanoparticles enhanced collagen I, osteocalcin expression and alkaline phosphatase activity in osteoblasts, indicating their potential to boost bone formation through improved cellular activities.

Although many papers present a large panel of in vitro tests covering several important aspects of understanding the bone formation process, the focus on certain markers, such as ALP, underscores their significance in evaluating osteogenic activity. While most in vitro studies in this review concluded that the presence of strontium associated with the biomaterial increased the expression/activity of ALP, Zhang et al. [64] found no difference compared to the control and its biomaterial releases up to 50 ppm in culture medium after 24 h, while Yang et al. [62] reported that the addition of Sr had an inhibitory effect on ALP. However, considering that the effect of strontium on osteoblastic cells is dose-dependent and that both studies did not evaluate the amount of strontium released from the biomaterials, it is possible that the inhibitory effects may be associated with a release of strontium ions at toxic concentrations.

The ALP activity in bone tissue is usually high. This protein plays an essential role in the modulation of bone tissue activity since it is responsible for providing phosphate ions to the formation of the mineralized extracellular matrix [71]. The high prevalence of articles that evaluate the effect of the biomaterial with strontium on this protein is due to its essential role in the formation of bone tissue. It occurs since ALP is an important confirmatory parameter that Sr is working on each biomaterial model tested. On the other hand, limiting the investigation to ALP does not add much to the understanding of the mechanisms of action of the ion on the cells/tissues. In fact, other proteins should be considered in future studies to elucidate the cellular response promoted by the presence of the biomaterial.

The cytotoxicity of strontium-doped biomaterials was less frequently reported. Hernandez et al. [27] concluded that the presence of strontium increased the cytotoxicity of the biomaterial compared with control. In that study, materials with the theoretical incorporation of strontium of 10 and 20% were analyzed, but no results were reported regarding the amount of this ion released, complicating the analysis of the possible causes of cytotoxicity. Since other studies with similar percentages of substitution have obtained divergent results [32], it is not possible to determine the molar ratio of calcium substitution by strontium that promotes in vitro toxicity.

Several studies performed both in vitro and in vivo tests, providing a more comprehensive understanding of the biomaterials’ performance. For example, Nguyen et al. [39] observed that titanium substrates coated with strontium-doped calcium phosphate ceramics exhibited enhanced cell adhesion and proliferation. This study emphasized the importance of optimizing the strontium doping concentration to achieve the desired cellular response and improve the biocompatibility of the coatings for bone regeneration applications. Olivier et al. [41] reported that strontium-doped bioceramics enhanced osteogenic differentiation and mineralization in mesenchymal stem cells. The study highlighted the importance of optimizing strontium concentrations to maximize the beneficial effects on cellular responses and improve the efficacy of biomaterials for bone tissue engineering.

In summary, the findings from in vitro studies consistently show that strontium incorporation into biomaterials enhances osteogenic differentiation, mineralization, and cellular proliferation, while maintaining favorable cytocompatibility. These improvements highlight the potential of Sr-doped biomaterials for various clinical applications in bone tissue engineering and orthopedic implants.

4.4. In Vivo Studies

Despite the need and search for alternative methods for animal use in science, in vivo tests are still regarded as important tools for the study of toxicity and efficacy of health products. From the references retrieved in the present review, all in vivo studies concluded that the addition of strontium to the biomaterial did not induce undesired toxic effects in the implant area, data that is in accordance with most in vitro results as discussed above. Gu et al. [23] observed a greater revascularization of the tissue after the implantation of the material containing strontium, which may indicate intense effects upon endothelial cells that could occur at systemic levels. However, since most in vivo studies did not evaluate the amount of strontium released from the biomaterial and none of them performed histologic labeling for cell death or inflammatory cells, it is not possible, at this time, to establish a relationship between the lack of inflammatory infiltrate and the presence of strontium.

One of the effects of strontium ranelate is to inhibit bone resorption by osteoclasts due to a decrease in RANK-L secretion, an essential factor for osteoclast differentiation and survival [72]. Similarly, Chung et al. [22] and Capuccini et al. [18] showed that the presence of strontium-doped calcium-phosphate biomaterials induce a decrease in osteoclastogenesis in vitro. However, the present search identified only one in vivo study demonstrating the presence of mature osteoclasts in direct contact with the biomaterial that could indicate active resorption performed by cells. Therefore, to clarify the physiological effects of strontium released from strontium-doped biomaterials in osteoclast function and metabolism, more studies in vitro and in vivo that access different recovery times after implantation are necessary.

In vivo studies also allow verification of the impact of ceramic ionic substitution in osteogenesis physiologically, with a focus on enhancing mechanical properties, osteogenic differentiation, and biocompatibility in different animal models and material types. Olivier et al. [41] demonstrated that strontium-doped bioceramics significantly enhanced osteogenic differentiation and mineralization in mesenchymal stem cells, indicating their potential for bone tissue engineering and highlighting the importance of optimizing strontium concentrations for therapeutic applications. Lourenço et al. [36] found that strontium-substituted hydroxyapatite coatings significantly enhanced bone-implant integration in rabbit models, supporting their potential for clinical applications in orthopedic and dental implants. Chen et al. [20] showed that strontium-doped mesoporous bioactive glass scaffolds significantly promoted bone regeneration in rat models with cranial defects, highlighting the dual osteogenic and angiogenic effects of strontium. Similarly, Chen et al. [21] observed that strontium-substituted hydroxyapatite coatings on polyetheretherketone (PEEK) implants significantly improved osseointegration and bone formation in rat femur defect models, supporting their potential for orthopedic applications. Harrison et al. [26] observed that strontium-doped bioceramics significantly enhanced the expression of osteogenic markers and promoted bone regeneration in similar rat calvarial defect models.

Interestingly, Li et al. [33] and Liang et al. [34] investigated different ranges of percentual substitution of calcium ions by strontium, and both concluded that theoretical substitution of 10% is the most favorable concentration to stimulate the formation of new bone. These data reinforce the hypothesis that the ideal amount of strontium released by the ceramic biomaterial must be within a safe range that unfortunately is still not completely determined.

The potential mechanisms through which strontium enhances bone regeneration include its impact on both angiogenesis and osteoclastogenesis. Zhao et al. [54] demonstrated that strontium incorporation into scaffolds significantly enhances bone regeneration in osteoporotic defects by modulating both phenomena. Similarly, Jiang et al. [31] demonstrated that hydroxyapatite bioceramics with micro-nano-hybrid surfaces and different strontium doping contents significantly promoted bone and blood vessel regeneration in rat models, finding a synergistic effect of surface modification and strontium doping on enhancing the osteogenic and angiogenic capacity of bioceramics. Ma et al. [37] also identified an enhancement of angiogenesis while investigating strontium-substituted hydroxyapatite coatings for polyethylene terephthalate (PET) artificial ligaments used in ACL reconstruction, in a rabbit model of anterior cruciate ligament reconstruction. The study revealed that the incorporation of strontium not only enhanced the mechanical properties of the implants but also led to improved ligament-bone integration by modulating angiogenesis and osteoclastogenesis.

The role of strontium in improving mechanical and physicochemical properties of CaPs was demonstrated by Pal et al. [42], who showed that Sr-doped hydroxyapatite derived from clam shells exhibited improved mechanical properties, making it a promising candidate for bone tissue engineering and in vivo applications. Ramadas et al. [43] further supported that Sr-substituted calcium phosphates improve mechanical strength and enhance osteogenic differentiation, showing significant improvements in mineral density and bone formation in rat femur defect models. Sun et al. [46] indicated that their newly developed Sr-containing hydroxyapatite bone cement exhibited improved compressive strength along with a favorable biocompatibility. Gu et al. [25] demonstrated that strontium-substituted hydroxyapatite coatings on magnesium alloys significantly controlled the degradation rate and enhanced bone regeneration in rabbit models. The study indicated that optimizing the Sr content in the coatings is crucial for balancing degradation behavior and osteogenic response in biomedical applications.

Overall, the reviewed studies provide robust in vivo evidence from animal models that the incorporation of strontium into various calcium phosphate-based biomaterials enhances bone regeneration and mechanical properties while maintaining favorable biocompatibility. These findings underscore the potential of Sr-doped biomaterials for clinical applications in bone repair and regeneration.

4.5. Involvement of Signaling Pathways

Smart materials can promote tissue support while stimulating beneficial responses such as lesion regeneration or infection reduction by activating or inhibiting several pathways and processes at the cellular level [73]. Understanding which intracellular signaling pathways are activated after contact with a strontium-functionalized ceramic biomaterial can help predict the behavior of the cell in response to the implant and, therefore, is an efficient way to understand how doping with strontium can help in the development of more efficient smart materials.

The Wnt/β-catenin pathway is crucial for bone tissue regeneration, as it plays a significant role in osteoblast differentiation and bone formation. Zarins et al. [63] showed that strontium incorporation enhances osteoblast differentiation and activity through the activation of the Wnt/β-catenin signaling pathway. This pathway’s activation promotes the expression of osteogenic markers and reduces the levels of pro-inflammatory cytokines such as TNF-α and IL-1β, creating a more favorable environment for bone regeneration. Similarly, Chen et al. [20] demonstrated that strontium-doped mesoporous bioactive glass scaffolds significantly promoted osteogenic differentiation and angiogenesis by activating the Wnt/β-catenin pathway. Gu et al. [25] also found that strontium-substituted hydroxyapatite coatings on magnesium alloys enhanced osteogenic differentiation through Wnt/β-catenin activation, further highlighting its importance in bone regeneration. Kozhemyakina et al. [74] emphasized the role of the Wnt/β-catenin pathway in sequential signaling for bone formation, highlighting the need for more studies exploring this pathway in the context of strontium-doped biomaterials.

The BMP-2 pathway is another critical regulator of bone formation, involved in the differentiation of mesenchymal stem cells into osteoblasts. Chen et al. [20] and Gu et al. [25] both reported that BMP-2 signaling is significantly enhanced by strontium-doped biomaterials, promoting osteogenic differentiation and mineralization. Ma et al. [37] confirmed these findings by showing that strontium-substituted hydroxyapatite coatings upregulated BMP-2 expression, along with other osteogenic and angiogenic markers, in rat bone marrow mesenchymal stem cells (rBMSCs). Jiang et al. [31] further demonstrated that strontium-doped hydroxyapatite bioceramics with micro-nano-hybrid surfaces significantly promoted the proliferation, differentiation, and angiogenic activity of bone marrow stromal cells (BMSCs) through the activation of BMP-2 and VEGF, directly related to the observed effects on bone tissue regeneration. BMP-2 initiates the process by signaling through the SMAD pathway, leading to the activation of Runx2, a key transcription factor in osteoblast differentiation. Birgani et al. [14] supported the role of BMP-2 in osteogenesis by demonstrating increased levels of this marker in the presence of strontium-doped materials.

Runx2 is a master transcription factor for osteoblast differentiation, and its activation is vital for bone tissue development. Xing et al. [50] demonstrated that air-plasma-treated titanium alloy coatings with strontium-doped phosphate significantly enhance osteogenic activity by upregulating Runx2. Sartoretto et al. [75] also found that SrCHA increased the expression of Runx2, Osterix, ALP, and collagen 1a1 genes, leading to higher osteogenic activity and bone formation. These findings underscore the importance of Runx2 in mediating the osteogenic effects of strontium-doped biomaterials. Brennan et al. [76] highlighted the critical role of Runx2 in initiating the mineralization process, which is supported by studies showing increased Runx2 expression in the presence of strontium-containing biomaterials [33,40,55].

The ERK pathway is known for its role in promoting osteogenic differentiation and angiogenesis, enhancing the expression of critical proteins like Runx2, BSP, and VEGF. Chen et al. [21] showed that strontium-substituted hydroxyapatite coatings on polyetheretherketone (PEEK) implants enhance osseointegration and bone formation through the activation of the ERK pathway. This pathway’s activation promotes the expression of osteogenic and angiogenic factors while simultaneously suppressing pathways such as p38, JNK, and AKT, involved in osteoclastogenesis, in a balance that is crucial for ensuring effective bone regeneration. Liu et al. [35] demonstrated increased secretion of growth factors such as bFGF and VEGF in vivo, particularly in the presence of ceramic material implants containing strontium, which supports the role of the ERK pathway in enhancing vascularization and bone regeneration.

Growth factors are of high relevance in bone metabolism and are also targets for stimulation during the development of smart materials. It has already been shown that the presence of strontium associated with calcium phosphate-based materials significantly increased the expression and release of bFGF and VEGF [23]. Liu et al. [35] demonstrated in vivo that there was an increase in the secretion of both growth factors, especially in the presence of the ceramic material implant containing 8% strontium substitution. These data make it possible to hypothesize that this type of material can increase the vascularization in the implant site, which is essential to the process of regeneration of any lesion. Huang et al. [28] proposed that the presence of a ceramic with 8% substitution of calcium ions by strontium can decrease TNF-α secretion while increasing OPG expression and decreasing RANK-L secretion. The combination of these factors could result, in vivo, in the reduction of inflammatory cells migration to the lesion site, thus accelerating the regeneration of the bone tissue. This hypothesis requires confirmation and the study of other inflammatory cytokines in addition to TNF-α, since Gu et al. [23] have shown that there is an increase in the expression of PECAM-1 in the presence of strontium. Due to the complexity of the bone tissue, the increase in the expression of this gene could be related to inflammatory signaling, promoting an increase in the population of leukocytes. It could also provide further evidence that Sr-doped calcium phosphates favor the formation of new blood vessels, which is essential to the process of injury regeneration [23].

The literature evidence indicates that incorporating strontium into calcium phosphate-based biomaterials holds significant potential for enhancing bone regeneration through the activation of key signaling pathways such as Wnt/β-catenin, BMP-2, Runx2, and ERK. These pathways play vital roles in regulating osteoblast differentiation, mineralization, and angiogenesis, essential for effective bone tissue regeneration. Future studies should continue exploring these pathways and their interactions to optimize the design and functionality of strontium-doped biomaterials for bone repair and regeneration.

In this systematic review, several limitations must be acknowledged. First, the search key was restrictive and may have missed studies involving more complex presentations of strontium-doped hydroxyapatite or other biomaterials. Second, the review focused primarily on in vitro and in vivo studies, potentially overlooking significant clinical correlations and results from human trials. Lastly, the limitation to complete reports might have excluded valuable data from published abstracts and other partial publications. Despite these limitations, the review provides substantial evidence supporting the positive effects of strontium incorporation into calcium phosphate-based biomaterials. Strontium has been shown to enhance osteogenic differentiation, mineralization, and cellular proliferation, while maintaining favorable cytocompatibility. Studies consistently demonstrated that strontium-doped materials significantly stimulate osteoblast activity, crucial for effective bone healing. Furthermore, strontium’s impact on osteoclast activity, reducing bone resorption, aligns with its known benefits in osteoporosis treatment, as summarized in Figure 3.

Figure 3. Schematic representation of the effects of doping calcium phosphate-based biomaterials with strontium ions on biological events, from strontium ion release into the biological medium to novel bone formation. Strontium ion release from the biomaterial [12,19,20,21,23,30,31,37,40,44,45,47,48,52,53,54,64] leads to preosteoblast proliferation [20,24,25,28,30,31,32,33,35,41,46,49,50,53,57] and differentiation into osteoblasts [11,15,18,21,22,30,36,47,53,54,57], with increased ALP activity [14,18,20,31,32,33,37,45,47,49,50,51,55,57], and expression of bFGF [35], BMP2 [27,31,55,63], BSP [14,31], OCN [14,17,18,55], OC [45,47,51,63] OPN [14,18,20,51], RUNX2 [20,33,55,57] and Type I collagen [16,18,20,31,36,45,51,55,63] Eventually, these cells turn into osteocytes, which produce mineralized tissue [36,37,43,49,50,57] and novel bone formation 18, 24, 33, 34, 36, 43, 49, 50, 51, 53, 54, 56, 61, 63]. Strontium also induces the release of angiogenic factors, enhancing tissue regeneration [25,31,35,43,48,54]. Additionally, its effects on monocytic cells through OPG [31,35,48,63], Trance [18] and RANKL [18,22,29,58] decrease osteoclastogenesis [18,21,22,29], with reduced Pit [62], TRAP [21,49], CAII [21] and osteoclast activity [18,49,62], resulting in lower bone resorption [58]. Together, these events contribute to novel bone formation. Arrows indicate molecules, genes, or events that are upregulated (↑) or downregulated (↓). Each number represents a reference providing evidence for the indicated event.

The literature, however, presents certain gaps. There is a notable lack of studies evaluating the exact release rates of strontium ions from biomaterials into biological media, complicating the establishment of safe and effective dosage ranges. Additionally, the specific signaling pathways through which strontium exerts its effects—such as Wnt/β-catenin, BMP-2, Runx2, and ERK—have not been comprehensively explored across all relevant studies. The understanding of how these pathways interact in the presence of strontium-doped biomaterials remains incomplete.

Future research should aim to address these gaps by investigating the release kinetics of strontium ions in various biological environments and focusing on the detailed mechanisms of cell signaling pathways activated by strontium. This would enable the optimization of strontium concentrations in biomaterials, ensuring both efficacy and safety. Such advancements could significantly improve the design and application of strontium-doped biomaterials for clinical use in bone repair and regeneration, ultimately leading to more effective and reliable treatment options for patients with bone-related conditions.

5. Conclusions

This systematic review aimed to investigate the biological effects of strontium-doped calcium phosphate biomaterials, focusing on their potential in bone therapy. The incorporation of strontium into calcium phosphate-based biomaterials has shown promising results in enhancing bone metabolism, osteoblast proliferation, and tissue regeneration. The review identified robust in vivo and in vitro evidence from various studies indicating that strontium-doped biomaterials can effectively stimulate osteoblast differentiation, proliferation, and new bone formation while inhibiting osteoclast activity, through the activation of key signaling pathways such as Wnt/β-catenin, BMP-2, Runx2, and ERK.

The results also highlight the importance of the release profile of strontium ions, which plays a critical role in determining the biocompatibility and therapeutic effectiveness of the biomaterials. Controlled and sustained release of strontium is essential for achieving the desired biological effects without causing toxicity. The studies reviewed showed a wide range of strontium release concentrations, from 0.5 ppm to over 20,000 ppm, indicating the need for careful optimization of the release kinetics to balance efficacy and safety, and revealing the dose-dependent nature of strontium’s biological effects. At lower concentrations (0.5% to 2.5%), strontium-doped biomaterials are non-cytotoxic and promote osteoblast activity and new bone formation. Moderate concentrations (2.5% to 5%) continue to support high osteoblast differentiation and proliferation with moderate osteoclast inhibition. However, higher concentrations (10% to 20%) exhibit increased cytotoxicity, reducing the overall efficacy of bone regeneration. This comprehensive analysis supports the potential of strontium-doped calcium phosphate biomaterials as a promising tool for bone tissue engineering and regenerative medicine.