Statins—Their Role in Bone Tissue Metabolism and Local Applications with Different Carriers

Statins, widely prescribed for lipid disorders, primarily target 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase competitively and reversibly, resulting in reduced low-density lipoprotein cholesterol (LDL-C). This mechanism proves effective in lowering the risk of lipid-related diseases such as ischemic cerebrovascular and coronary artery diseases. Beyond their established use, statins are under scrutiny for potential applications in treating bone diseases. The focus of research centers mainly on simvastatin, a lipophilic statin demonstrating efficacy in preventing osteoporosis and aiding in fracture and bone defect healing. Notably, these effects manifest at elevated doses (20 mg/kg/day) of statins, posing challenges for systematic administration due to their limited bone affinity. Current investigations explore intraosseous statin delivery facilitated by specialized carriers. This paper outlines various carrier types, characterizing their structures and underscoring various statins’ potential as local treatments for bone diseases.


Introduction
Statins, originally derived from mold fungi, constitute a class of medications widely employed in treating lipid disorders, which stand as a primary risk factor for atherosclerosis and related conditions like coronary artery disease, ischemic cerebrovascular disease, and peripheral vascular diseases [1][2][3].These compounds function as competitive, reversible inhibitors of the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, a key catalyst in the early stages of hepatic cholesterol biosynthesis.Approximately two thirds of the body's cholesterol is synthesized de novo in the liver, with the transformation mediated by HMG-CoA reductase serving as the rate-limiting step in this process [4].
Inhibiting this enzyme's activity diminishes cellular cholesterol levels, prompting the upregulation of low-density lipoprotein (LDL) receptors on the cell surface and enhancing the cellular uptake of LDL from the bloodstream.The consequential reduction in plasma LDL stands as the primary therapeutic effect of statins [4,5].Additionally, these drugs exhibit the capability to modestly decrease triglyceride levels, elevate high-density lipoprotein (HDL) concentration, and reduce lipid susceptibility to oxidation [6].Moreover, their cardioprotective effects may extend beyond lipid profile modulation, possibly involving increased endothelial nitric oxide synthesis, leading to improved vascular endothelial function [7].
Statins might mitigate cardiovascular event risk by stabilizing atherosclerotic plaques, inhibiting metalloproteinase secretion by macrophages, and impeding extracellular matrix breakdown, thus fortifying plaque fibrous membranes [8,9].This drug class also influences the coagulation system, diminishing platelet aggregation and promoting fibrinolysis [6,10].Statins may further modulate arterial wall cell numbers by restricting smooth muscle cell proliferation and enhancing apoptosis [11].Notably, they exhibit antioxidant, immunomodulatory, and anti-inflammatory effects, addressing the emerging emphasis on chronic inflammation's role in atherosclerosis development [12,13].Their pleiotropic effects and generally favorable patient tolerance make statins a cornerstone in cardiovascular disease treatment (see Figure 1).fibrinolysis [6,10].Statins may further modulate arterial wall cell numbers by restricting smooth muscle cell proliferation and enhancing apoptosis [11].Notably, they exhibit antioxidant, immunomodulatory, and anti-inflammatory effects, addressing the emerging emphasis on chronic inflammation's role in atherosclerosis development [12,13].Their pleiotropic effects and generally favorable patient tolerance make statins a cornerstone in cardiovascular disease treatment (see Figure 1).Commonly utilized statins include mevastatin, lovastatin, and pravastatin (natural statins); simvastatin (a semi-synthetic statin); and fluvastatin, rosuvastatin, pitavastatin, cerivastatin, and atorvastatin (synthetic statins) [14,15].Their chemical formulas are depicted in Figure 2. Notably, variations in their chemical structures impact lipophilicity, hydrophilicity, and subsequent absorption, distribution, metabolism, and excretion [14,16].
The molecule of each statin consists of three main parts: an HMG-CoA analogue, a complex ring structure responsible for binding the statin to HMG-CoA reductase, and a side chain structure determining solubility.Statins vary greatly in solubility due to the presence/absence of polar moieties in their predominantly hydrophobic backbones.Lipophilic statins can easily penetrate deeper into cell membranes and enter cells through passive diffusion, and are therefore widely distributed in various tissues.They are metabolized by cytochrome P450 (CYP) enzymes upon binding to the membrane.In turn, hydrophilic statins remain bound to the polar surface of the membrane and require the transport of proteins into the cell, show greater hepatoselectivity and lower potential for uptake by peripheral cells than lipophilic statins, and are mostly eliminated unmetabolized.
The molecule of each statin consists of three main parts: an HMG-CoA analogue, a complex ring structure responsible for binding the statin to HMG-CoA reductase, and a side chain structure determining solubility.Statins vary greatly in solubility due to the presence/absence of polar moieties in their predominantly hydrophobic backbones.Lipophilic statins can easily penetrate deeper into cell membranes and enter cells through passive diffusion, and are therefore widely distributed in various tissues.They are metabolized by cytochrome P450 (CYP) enzymes upon binding to the membrane.In turn, hydrophilic statins remain bound to the polar surface of the membrane and require the transport of proteins into the cell, show greater hepatoselectivity and lower potential for uptake by peripheral cells than lipophilic statins, and are mostly eliminated unmetabolized.
Upon oral administration, statins undergo substantial hepatic first-pass effects, limiting their systemic bioavailability to 5-30% of the administered dose due to high hepatic uptake.In plasma, nearly 95% of statins and their metabolites are protein-bound [14,16].Some, like simvastatin, are administered as inactive prodrugs requiring hydrolysis catalyzed by esterases and peroxidases for therapeutic effect.Despite generally good tolerability, serious adverse effects, especially at higher doses, may occur.Serving as substrates for CYP3A4 (cytochrome P450 family 3 subfamily A member 4) isoenzyme and OATP-2 (organic anion transporting polypeptide-2), statins can engage in clinically significant interactions with other drugs affecting these proteins.
(organic anion transporting polypeptide-2), statins can engage in clinically significant interactions with other drugs affecting these proteins.Common adverse effects of statin therapy include elevated liver transaminases, with hepatotoxicity and nephrotoxicity as potential risks.Some rare but severe adverse effects are myopathy and rhabdomyolysis, with the risk directly proportional to statin concentration in plasma [17,18].
Currently, research explores statin use beyond cardiac diseases related to lipid disorders, including potential applications in bone diseases, injuries, and tissue defects [19,20].
Our study focuses on investigating the effects of statins on bone tissue metabolism, considering different carriers for drug application.
It should be noted that statins, like most drugs, are typically administered orally.However, the oral administration of statins presents drawbacks, such as first-pass metabolism in the liver and degradation in the gastrointestinal tract, resulting in limited bioavailability.Another concern is the occurrence of adverse effects such as myopathy, kidney and liver damage, and rhabdomyolysis.Hence, alternative routes of statin administration are under investigation, particularly for potential use in bone diseases where prolonged action directly within the bone tissue is required.
We conducted a comprehensive search on databases such as PubMed, Embase, IEEE Explorer, and Google Scholar, accessing them on 27 December 2023, due to their leading role in scientific research and English-language content.The search phrases included "statin bone tissue," "statin bone metabolism," "statin bone application," "statin bone," "statin osteoporosis," and "statin biomaterials."The publications considered spanned from 1999 to 2023, complemented by insights from pharmacology and pharmaceutical chemistry books, contributing approximately 15% of the overall information.Common adverse effects of statin therapy include elevated liver transaminases, with hepatotoxicity and nephrotoxicity as potential risks.Some rare but severe adverse effects are myopathy and rhabdomyolysis, with the risk directly proportional to statin concentration in plasma [17,18].
Currently, research explores statin use beyond cardiac diseases related to lipid disorders, including potential applications in bone diseases, injuries, and tissue defects [19,20].
Our study focuses on investigating the effects of statins on bone tissue metabolism, considering different carriers for drug application.
It should be noted that statins, like most drugs, are typically administered orally.However, the oral administration of statins presents drawbacks, such as first-pass metabolism in the liver and degradation in the gastrointestinal tract, resulting in limited bioavailability.Another concern is the occurrence of adverse effects such as myopathy, kidney and liver damage, and rhabdomyolysis.Hence, alternative routes of statin administration are under investigation, particularly for potential use in bone diseases where prolonged action directly within the bone tissue is required.
We conducted a comprehensive search on databases such as PubMed, Embase, IEEE Explorer, and Google Scholar, accessing them on 27 December 2023, due to their leading role in scientific research and English-language content.The search phrases included "statin bone tissue", "statin bone metabolism", "statin bone application", "statin bone", "statin osteoporosis", and "statin biomaterials".The publications considered spanned from 1999 to 2023, complemented by insights from pharmacology and pharmaceutical chemistry books, contributing approximately 15% of the overall information.

Statins-Their Role in Bone Tissue Metabolism
As early as the late 20th century, observations indicated that statins can stimulate osteogenesis and offer therapeutic benefits to patients with osteoporosis [21][22][23][24][25]. Subsequent research has unveiled their potential as robust modulators of bone healing and the inflammation process, demonstrating their capacity to stimulate osteoinduction and angiogenesis-key phenomena for bone reconstruction [26,27].They also exhibit the ability to limit the loss of bone tissue by inhibiting both osteoclastogenesis and the apoptosis of osteoblasts [28][29][30].Therefore, the beneficial effect of statins on bone tissue metabolism results from their action through various mechanisms, as confirmed in numerous animal models of osteoporosis and mechanical bone damage [31][32][33][34][35]. Lipophilic statins, particularly simvastatin, lovastatin, and atorvastatin, appear to be the most effective in preventing osteoporosis and supporting the healing of bone fractures and defects [22,23,36].
Several mechanisms have been proposed to explain the anabolic effect of statins on bone tissue (see Figure 3).Firstly, these drugs can stimulate the formation of new tissue by increasing the expression of a gene for bone morphogenetic proteins (BMPs), specific growth factors regulating osteoblast and chondroblast proliferation and differentiation [21,26,37,38].Statins increase BMP-2 expression by modulating small GTP-binding proteins, particularly the Ras and Rho proteins [37].It is important to note that only lipophilic statins can activate the promoter of this gene.Another suggested mechanism related to the primary target point of statins involves blocking HMG-CoA reductase, inhibiting the conversion of HMG-CoA to mevalonate, and indirectly limiting the biosynthesis of its derivatives, such as farnesyl pyrophosphate or geranylgeranyl pyrophosphate [22,[36][37][38][39].These derivatives adversely affect bone tissue metabolism.For example, farnesyl diphosphate can activate the glucocorticoid receptor, potentially increasing bone resorption and reducing osteoblast proliferation.Geranylgeranyl pyrophosphate likely also negatively affects osteogenesis [21,22].By affecting the TGFβ/Smad3 pathway, statins protect osteoblasts against apoptosis.TGFβ (transforming growth factor beta) activates Smad3, promoting the formation of new bone tissue by stimulating the synthesis of extracellular matrix proteins, intensifying mineralization, and increasing alkaline phosphatase activity-a marker of early osteoblast differentiation (see Figure 3) [28,30].In studies on a mouse osteocyte cell line, these drugs have been shown to increase the expression of the OPG gene and decrease the expression of the RANKL gene [22,24].It has also been proven that the number of osteoclasts decreases with increasing statin concentration.Studies also suggest that statins may promote neovascularization because they increase gene expression for vascular endothelial growth factor (VEGF) and fibro- Studies have shown that bone marrow stem cells in the presence of statins exhibit increased activity of this protein, with the drug released from the carrier stimulating the mineralization of the extracellular matrix [40][41][42][43].The loss of Smad3 results in the apoptosis of osteoblasts and osteocytes, leading to decreased bone mass.By enhancing its expression in these cells, statins may contribute to their protection.These drugs can also modulate osteoclastogenesis by inhibiting it through the OPG/RANKL/RANK pathway [40].RANKL (receptor activator of NF-κB ligand), RANK (receptor activator of NF-κB), and osteoprotegerin (OPG) play key roles in this process.OPG reduces the proliferation of osteoclasts, while RANK, belonging to the tumor necrosis factor receptor (TNFR) superfamily, enhances osteoclastogenesis.RANKL, being a membrane protein on the surface of osteoblasts, acts as a key factor for osteoclast function by blocking their apoptosis [26].Mice lacking RANKL or RANK developed significant osteoporosis due to the complete absence of osteoclasts and the consequent lack of bone resorption and remodeling.In turn, OPG-deficient mice showed osteoporosis resulting from an increased number of osteoclasts.The balance of this signaling pathway is crucial for proper, balanced bone remodeling, and statins can modulate it.
In studies on a mouse osteocyte cell line, these drugs have been shown to increase the expression of the OPG gene and decrease the expression of the RANKL gene [22,24].It has also been proven that the number of osteoclasts decreases with increasing statin concentration.Studies also suggest that statins may promote neovascularization because they increase gene expression for vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF-2).The processes of osteogenesis and angiogenesis are closely related, and coordinated vascularization is of key importance in the process of bone tissue reconstruction.It not only provides oxygen and nutrients to the developing tissue, but also allows the transport of mesenchymal stem cells from the bone marrow, further supporting bone regeneration [44][45][46][47][48][49].
The ability of statins to simultaneously stimulate these two processes makes them even more promising potential drugs in the therapy of bone damage and defects [50,51].Unfortunately, systemically administered statins have a low affinity for bone tissue, making the systemic use of these drugs in attempts to treat bone diseases potentially inconclusive as to their effectiveness [51,52].Additionally, as mentioned previously, most statins undergo a significant first-pass effect, further limiting their bioavailability in the bones.Therefore, orally administered statins in clinical doses required for lipid-lowering therapy are not able to exert a strong positive effect on the anabolism and catabolism of bone tissue [51][52][53].Achieving these effects would require administering drugs at much higher doses, significantly increasing the risk of systemic adverse effects.Therefore, the topical application of statins directly to the bone tissue may be an alternative option that allows for bypassing the hepatic metabolism of these drugs, increasing their bioavailability, and reducing the dose.Other resulting benefits include reducing the risk of systemic adverse effects, as well as eliminating the need for repeated administration of the drug.

Statin Local Delivery Methods and Carriers
The local administration of statins in the case of bone tissue disorders seems justified for several reasons.Firstly, bone tissue is highly vascularized, and systemic treatment requires the use of large doses of therapeutic substances to achieve an adequate concentration at the target site [54,55].Therefore, the direct administration of statins to bone tissue allows for a reduction in dosage compared to systemic administration [56].Additionally, the carrier for intraosseous drug delivery can provide a matrix for the infiltration of mesenchymal cells and often serves as a filling material for bone defects [57,58].It is essential to emphasize that the optimal carrier should exhibit appropriate degradation rates to ensure the proper growth of newly formed bone tissue while preventing the formation of fibrous tissue and fibrous encapsulation of the carrier.The local administration of statins can effectively reduce the risk of adverse effects, primarily myopathy characterized by muscle pain and weakness, the elevation of creatine kinase (CK) levels in the serum, and, in extreme cases, rhabdomyolysis, which is a life-threatening condition [57,58].
It is worth noting that our study did not focus on the specific doses of statins used in individual research.The results regarding statin dosage in the treatment of bone tissue diseases remain inconclusive.Depending on the dose, these drugs can exhibit either positive or negative effects on bone health.For instance, high doses of simvastatin (20 mg/kg bw/day) have been shown to stimulate new bone tissue formation, while low doses (1 mg/kg bw/day) may inhibit reconstruction and increase bone resorption [59].Despite promising results, the practical applications of statins in treating bone diseases require further research, including optimizing dosage and exploring effective methods of application.
Carriers were equipped with statins in various ways.The simplest method involves impregnating bone scaffolds, but this has the drawback of rapid drug dissolution in the bloodstream, leading to reduced bioavailability.Alternatively, spray techniques and ultrasonic dispersion can coat statins onto bone scaffolds, allowing for only low-dose application.Other approaches involve micro-and nanospheres, chemical compound combinations, polymerization, and blending with biomaterials, each with its advantages and limitations.
Based on their chemical composition, carriers can be categorized into those made of inorganic materials, natural polymers, synthetic polymers, and polymer/inorganic material composites.The choice of carrier materials is dictated by the structure and properties of bone tissue, composed of an organic matrix (mainly collagen type I fibers) and an inorganic phase, primarily biological apatite, a nanocrystalline calcium phosphate.Inorganic carriers offer high compatibility, bioactivity, and bioresorbability, but may lack sufficient mechanical strength, especially when serving as bone defect fillers.On the other hand, polymers, despite high biocompatibility, may lack the necessary hardness.Therefore, composite carriers seem to be the optimal solution, providing adequate mechanical properties, biodegradation, and controlled statin release at the target site.
In the case of inorganic carriers, calcium phosphates are primarily utilized due to their similarity to biological apatite.Among the most commonly used calcium phosphates, hydroxyapatite and crystalline calcium orthophosphates (in both low-and high-temperature forms; respectively, β and α) can be mentioned, with α-TCP being a compound that is much more soluble than β-TCP and hydroxyapatite.α-TCP has been employed in the form of bone cement, a material that solidifies in reaction with water to form a hard filling for bone defects.β-TCP has also been used in the form of cement, as well as a biphasic material with hydroxyapatite, thereby increasing the material's bioresorbability and bioactivity.In the study [72], unconventional hydroxyapatite in the form of nanofibers was used, while in [73], together with simvastatin, it served as a coating for a titanium implant.In this case, hydroxyapatite was used as a factor for improving osteointegration and as a carrier for delivering a statin.The studies [74,75] focus on the use of a scaffold of calcium sulfate loaded with simvastatin.Research conducted using inorganic materials was carried out in both in vitro conditions (primarily on BMSCs cells) and in vivo animal models (rats, mice, and rabbits).In all the presented studies, the simultaneous administration of the inorganic material with statin resulted in the increased formation of new bone tissue, increased density of the created tissue, and improvement in bone tissue regeneration, which were observed between the 3rd and 12th week of the experiment.
Significantly more research has focused on the utilization of both natural and synthetic polymers, as well as composite materials, as potential carriers for statins.This emphasis likely stems from the inherent drawbacks of inorganic materials (primarily calcium phosphates), notably, their brittleness and, thus, insufficient mechanical strength, which render them unsuitable for areas subjected to high stresses.Polymers are characterized by high flexibility, whereas calcium phosphate/polymer composites combine the hardness of inorganic materials with the elasticity of polymers, ensuring excellent mechanical properties akin to those of bone tissue, which is also a natural composite.
Among the natural polymers utilized for statin carriers, chitosan has been predominant, with collagen or prepared atelocollagen being alternative materials.Gelatin has also been employed in hydrogel form.Concerning synthetic polymers, a wide array have been utilized, including both biodegradable (e.g., PLA, PLLA, PCL, PLGA) and nonbiodegradable (PUR, PEEK) polymers.The composite materials described in the available literature consist of hydroxyapatite or beta-TCP combined with polymers such as poly(ecaprolactone), poly(glycerol sebacate), gelatin-nanofibrillar cellulose, PCL, and PLGA.Polymer and composite materials are prepared in various forms, including porous scaffolds, microspheres, nanomicelles, fibers, nanoparticles, and coatings on titanium surfaces.The available literature predominantly comprises in vivo studies on rats and rabbits, which consistently indicate increased mineralization, bone tissue formation, and improvements in bone parameters in each case.
A significant aspect investigated under laboratory conditions is the release of statins from previously obtained carriers.Various release profiles have been observed depending on the chemical composition of the carrier, its form, and its preparation method.Generally, the constructed carriers are designed to release statins gradually over an extended period (from 50-90% within 7 days) [76][77][78].In the study by [78], a typical microporous structure of calcium phosphate foam was employed, which proved to be a suitable carrier for pitavastatin: the "burst release" effect was relatively minimal, followed by the gradual release of the statin over 72 h.Various approaches have been applied to prolong the release time.For instance, delayed release was achieved in carriers derived from biomimetic beta-TCP by coating it with an additional layer of apatite [63].This resulted in a matrix that released 20% less simvastatin over 7 days.In the study by [79], significantly prolonged release of simvastatin was obtained from PCL microspheres contained within a collagen coating covering PET polymer (constructing artificial ligaments).Over 14 days, slightly over 50% of the drug was released, and approximately 75% within nearly 40 days.

Conclusions
The treatment of large bone defects and effective bone tissue regeneration remain challenges in orthopedics and dental surgery.Constant efforts are made to identify both bone substitute materials with appropriate physicochemical, mechanical, and biological properties, as well as factors that would expedite the process of new bone tissue formation and the development of blood vessels within it.
Therefore, the discovery that statins, drugs that have long been used to lower cholesterol levels in the blood, exhibit strong proangiogenic and osteogenic effects has generated significant interest in their potential use as supportive factors in the treatment of serious bone disorders.Our literature review has demonstrated that statins, acting pleiotropically through various mechanisms, attenuate osteoblast apoptosis, influence the reduction in osteoclast differentiation, and stimulate bone tissue formation, among other effects, by promoting osteoblast differentiation.
Of course, like most drugs, statins exhibit numerous adverse effects, which can be particularly hazardous when high doses are required for the therapy of bone disorders.Hence, much research is focused on developing local delivery systems for statins to bone tissue, which, in addition to minimizing adverse effects, would improve bioavailability and allow for a reduction in drug dosage.
The results of this review suggest that carriers based on inorganic materials (primarily various calcium phosphates and calcium sulfate), polymeric materials (especially chitosan, collagen poly-e-caprolactone, and poly(lactide-co-glycolide)), and inorganic-organic composite materials are capable of delivering statins locally to bone tissue, releasing them over time, and significantly enhancing bone tissue regeneration compared to mere filling.The findings from these studies are promising and highlight the need for further investigation.It is worth noting that only one human study has been published so far; thus, clinical trials should proceed.
While there are numerous works in the literature dedicated to the issue of bone carriers for statins, there are no articles focusing on the combination of different antiresorptive and osteogenic factors with statins.Future research could be dedicated to examining the impact of the simultaneous administration of statins and other factors stimulating bone tissue regeneration (e.g., magnesium ions, strontium, or silicates).These could be introduced in the form of substituted calcium phosphate.Future studies should also focus on determining the appropriate statin dosage under in vivo conditions to ensure therapy effectiveness and safety.It is worth noting that the majority of studies only address one statin, simvastatin.Therefore, further research should also include other representatives of this group.

Table 1 .
The effects of various statins combined with different types of carriers on bone.