An Exploration of the Role of Osteoclast Lineage Cells in Bone Tissue Engineering

Inhibitory soluble factors

BTE constructs can be loaded with soluble factors that can be released to surrounding tissue over a controllable time scale, where the released factor can focus its effect at the implantation site. Numerous studies have shown the possibility of inhibiting OcLCs with specifically drug-laden BTE constructs (Table 2). ⁴⁶ Many of these drugs are indicated for osteoporosis and act to promote bone formation by inhibiting OcLCs. ⁴⁷ For example, bisphosphonates such as alendronate are commonly prescribed medications for osteoporosis that inhibit osteoclastogenesis. ⁴⁸ Zeng et al. capitalized on this by creating graphene oxide-related hydrogels and incorporating them into a Type 1 collagen sponge loaded with alendronate. ⁴⁹ In vivo studies revealed that alendronate-containing sponges implanted into bony defects in a rat model were associated with a decreased osteoclast number and suppression of systemic bone loss as compared with alendronate-free sponges. Alternatively, Posadowska et al. developed an injectable system of alendronate-loaded nanoparticles separately suspended in a gellan gum (GG) hydrogel encapsulated in poly(lactide-coglycolide) (PLGA). ⁵⁰ This system was associated with suppressed osteoclastogenesis of RAW264.7 cells, even despite RANKL administration. Yet they underwent robust osteoclastogenesis without the system. It is also noteworthy that this system was cytocompatible with osteoblast-like cells, suggesting potential for regenerative applications. Another notable bisphosphonate is clodronate. Hayden et al. compared the effect of alendronate to clodronate on osteoclasts, osteoblasts, and a coculture of the two over 12 weeks on a silk-HA film. ⁵¹ Both drugs could induce beyond a 90% reduction in osteoclast metabolic activity by the fifth day of culture. However, high doses of clodronate were observed to preserve the metabolic activity of osteoblasts when compared with alendronate, indicating potential utility in bone regeneration. Alongside well characterized effects, these works establish bisphosphonates as a potentially potent means of promoting bone regeneration partly through OcLC inhibition.^52–53

Other drugs outside the bisphosphonate class have been used to modulate OcLCs. Zhang et al. demonstrated a synergistic effect of resveratrol, a polyphenolic photoalexin derived from various plants, and strontium (Sr) ranelate, a medication for osteoporosis, on bone regeneration. ⁵⁴ This involved 3D printing of scaffolds consisting of polycaprolactone (PCL), β-TCP, and a hydrogel-based bioink. The scaffolds were loaded with resveratrol, Sr ranelate, or both and evaluated for their influence on osteoclast activity. The group observed that the release of both components and Sr ranelate only decreased osteoclast size and tartrate-resistant acid phosphatase (TRAP) activity when compared with resveratrol only and a scaffold only control. They also observed that this dual delivery was associated with promotion of osteoblastogenesis and in vivo bone formation beyond scaffold only and defect only controls. Drugs with applications outside bone diseases have also been used. Petit et al. explored using thermosensitive chitosan hydrogels functionalized with D-α-tocopherol polyethylene glycol succinate-based nanoemulsions. ⁵⁵ These hydrogels contained atorvastatin or lovastatin, common statins used to prevent cardiovascular disease and hyperlipidemia, to modulate bone cells pleiotropically. An in vitro experiment involved infection of TERT-2 OKF-6 human oral epithelial cells and human oral fibroblasts with Porphyromonas gingivalis to assess for inflammatory and osteoclastic markers. They observed that treatment with atorvastatin-loaded or lovastatin-loaded nanoemulsions led to reduced expression of RANKL as compared with the absence of statins. They also observed the nanoemulsions to be associated with increased expression of bone sialoprotein 2 (BSP2) in osteoblasts beyond untreated cells and systemic statin administration. Furthermore, in vivo experiments revealed that treatment of calvarial bone defects with these hydrogels was associated with increases in new bone formation as compared with untreated and statin-free hydrogel controls, as well as systemic statin administration. Interestingly, Pan et al. used cabozantinib, a cancer drug, in a hyaluronic acid-based hydrogel. ⁵⁶ They assessed it in preosteoblasts and human bone-derived 786-O (Bo-786) renal cell carcinoma cell cultures and in a mouse model of renal cell carcinoma femoral bone metastasis. Cabozantinib led to a reduction in TRAP-positive multinuclear cells, hallmark features of mature osteoclasts, that formed in vitro, in comparison with a cabozantinib-free condition. It also was associated with enhanced preosteoblast differentiation despite the presence of Bo-786 cells, as evidenced by increases in alkaline phosphatase and osteocalcin expression beyond controls. Furthermore, the in vivo experiment demonstrated a reduced osteoclast number, increased osteoblast number, and increased bone volume in response to the hydrogel system when compared with controls. Gallium nitrate is another oncological drug used to treat hypercalcemia secondary to cancer through osteoclast inhibition. Ghanta et al. evaluated the ability of gallium acetylacetone (GaAcAc) released from methylcellulose-based hydrogels to suppress osteoclastogenesis of RAW264.7 or hematopoietic stem cells. ⁵⁷ They observed greater efficacy of these hydrogels as compared with the GaAcAc solution in reducing the degree of bone resorption observed in ex vivo experiments. These findings further corroborate the observation that drugs investigated up to the present have largely inhibitory effects on OcLCs. However, further work is needed to verify the consistency of these findings, and more drugs ought to be explored.

Various proteins and peptides have been observed to strongly influence the fate of OcLCs, as well as the broader bone remodeling system. ⁵⁸ This has been leveraged in BTE constructs with modulatory potential. BMP-2, a well-known osteogenic factor, has shown pro-osteoclastogenic and regenerative properties in multiple studies. However, excessive use of BMP-2 can induce harmful side effects. ⁵⁹ To address this, Arai et al. evaluated the supplemental local application of OP3-4 peptide, an analog of OPG, to promote bone formation in a murine tooth extraction model with a very low dose of BMP-2. ⁶⁰ A gelatin hydrogel-containing BMP-2 with or without OP3-4 peptide was implanted within the socket of a mandibular incisor of mice. New bone formation was most enhanced, and osteoclast numbers were lowered in the socket loaded with BMP-2 and OP3-4. Similarly, Wang et al. loaded a thermosensitive hydrogel with both BMP-2 and OPG to be evaluated with bone marrow MSCs (BMSCs) in vitro and in a rabbit model. ⁶¹ They observed that hydrogels that included OPG could reduce the expression of RANKL beyond those without it. They also observed that the combination of both proteins promoted osteoblastogenesis and new bone growth beyond either protein alone or controls. Altogether, while findings discussed in 4.2.1 display a pro-osteoclast and regenerative effect of BMP-2, these investigations here suggest that if bone regeneration is augmented with OPG or OP3-4, the pro-osteoclast property of BMP-2 may be overridden. Nevertheless, OP3-4 has also been investigated for BTE in isolation. Luo et al. incorporated it into gelatin methacrylate and evaluated it in a rat model. ⁶² They similarly observed significantly reduced osteoclast numbers and increased expression of osteogenic proteins with OP3-4 when compared with a pure gelatin methacrylate control. Lastly, certain synthetic peptides outside the typical discussion of bone biology have also been explored. Hu et al. explored using chitosan methacryloyl hydrogels loaded with human beta-defensin 1 short motif Pep-B in periodontal pockets. ⁶³ They observed that injection of this peptide-containing hydrogel significantly reduced osteoclast formation and alveolar bone loss. This further establishes the premise that certain proteins and peptides have demonstrated efficacy in inhibiting OcLCs as a mechanism of bone regeneration. However, more work is needed on what additional proteins and peptides can work through this mechanism outside of OPG and OP3-4 and how this can be applied in BTE.

Inhibitory environmental factors

Substantial work has revealed the potential of environmental factors to inhibit OcLCs in BTE, including ceramics, scaffold properties, plant-derived components, and nano-scale components (Table 3). Concerning ceramics, the bone matrix consists of organic components (collagen type I, proteoglycans, and glycoproteins) and inorganic components primarily in the form of HA. ⁶⁴ The latter has been extensively investigated for OcLC inhibition. Although studies discussed in 4.2.2 suggest an almost exclusively pro-osteoclast effect of HA, select investigators have observed contrasting results. Hadi et al. explored the use of carbonated HA-chitosan (CHA-CS) hydrogel derived from blood cockle shells in orthodontic relapse within a rat model. ⁶⁵ They observed a significant decrease in osteoclasts, increase in osteoblasts, and reduced relapse distance with CHA-CS when compared with controls. Furthermore, as discussed in 4.2.2, calcium-based ceramics in general exhibit largely pro-osteoclast and regenerative properties. However, certain noncalcium-based ceramic components have demonstrated the inverse effect on osteoclasts in direct comparisons with calcium-based ceramic components. Zhang et al. investigated the use of alginate microgels cross-linked with either Ca²⁺ or Sr²⁺ both in vitro and in a rat model. ⁶⁶ The Ca²⁺ condition exhibited significantly greater markers of bone regeneration in vitro, which the authors attributed to the association of Sr²⁺ with osteoclast inhibition. However, it should be noted that bone healing within the rat model was comparable between the two conditions. Noncalcium-based ceramics have also been evaluated in association with calcium-based ceramics. Panzavolta et al. prepared gelatin scaffolds with Sr substituted HA, which were associated with inhibited osteoclastogenesis and stimulation of osteoblasts beyond pure-HA scaffolds. ⁶⁷ While ceramics relevant to BTE tend to be osteoclastogenic, the former studies demonstrate interesting examples to the contrary. Further work is needed on alternative ways through which noncalcium-based ceramics like those involving strontium can support bone regeneration through this avenue. In addition, the unusual results with calcium-based ceramics seen with Hadi et al. warrant future exploration of the circumstances in which those results are probable and how this can be leveraged.

Select investigators have explored the manipulation of scaffold properties. For instance, Halai et al. explored micropatterning of a ceramic implant material in an MSC and bone marrow-derived hematopoietic cell coculture. ⁶⁸ Thirty micrometer diameter pits were associated with enhanced osteogenesis, reduced osteoclastogenesis, and a lower osteoclastic cell number than planar controls. This lends important insights into scaffold surface design considerations that ought to be further explored as a variable distinct from composition. Some groups have also explored plant-derived compounds, such as Bose et al., who investigated the effect of garlic extract within human MSC-monocyte cocultures seeded on calcium phosphate scaffolds. ⁶⁹ The extract was observed to reduce osteoclast resorption potential after 21 days and to increase the osteoblastic markers of osteocalcin by 1.6-fold and alkaline phosphatase by threefold after 14 days as compared with the control. It was also observed to enhance collagen formation within a rat model. Interestingly, Yao et al. examined the effect of traditional Chinese medicines, including Eucommia Oliv., Dipsacus asper Wall., Loranthus parasiticus Merr., Achyranthes bidentata Bl., and Cuscuta chinensis Lam. ⁷⁰ They incorporated them into gelatin and TCP-based composites that were placed in the parietal bone of neonatal rat calvarias organs. They observed that C. chinensis Lam. was associated with stimulation of osteoblastogenesis and decreased osteoclastogenesis when compared with Chinese medicine-free controls. While these works show a compelling outlook for plant-derived compounds as OcLC inhibitors in BTE, future studies should focus on further categorizing them in a way that enables better functional predictions.

A few groups have explored nano-scale inhibitory components. Shan et al. developed a hyaluronic acid-based hydrogel loaded with tobacco mosaic virus (TMV) nanoparticles and assessed their influence on osteoclastogenesis. ⁷¹ These TMVs were associated with reduced expression of osteoclastogenic genes when compared with a TMV-free control in vitro. They were also associated with enhanced bone mass when compared with pure hydrogel and a treatment-free control within a mouse tibial defect model. In the same study, this group also evaluated an RGD1 mutant variation TMV that displayed even greater potential in osteoclastogenesis inhibition. Moreover, Byun et al. infused tannic acid-mineral nanoparticles into a gelatin-based cryogel to evaluate their bone-forming potential. ⁷² After seeding RAW264.7 cells, nanoparticle-containing gels displayed lower levels of osteoclastogenic markers than controls. Furthermore, in vivo studies demonstrated that these gels were associated with new bone of higher quantity and quality than controls. Lastly, Li et al. evaluated zinc oxide and minocycline serum albumin nanoparticles for treating peri-implantitis. ⁷³ Compared with untreated and pure minocycline controls, hydrogels containing zinc oxide were observed to reduce osteoclast number and maintain supporting bone tissue within a murine disease model. This helps establish nano-scale components as an effective way to inhibit OcLCs in BTE, but more work is needed in characterizing their varying effects.

Inhibitory cellular factors

Another approach to OcLC inhibition is capitalizing on cell–cell interactions, such as signaling from MSCs (Table 4). ⁷⁴ For instance, Tcacencu et al. implanted a MSC-laden hydrogel into a bony defect within the mandible of a rat model and observed significant increases in bone volume at 1 week and reduced osteoclast number at 1 and 4 weeks as compared with cell-free controls. ⁷⁵ These results indicate MSCs may have efficacy in supporting bone regeneration through OcLC inhibition, but further investigation is needed to determine the consistency in which this holds true across different contexts. Nonbone cells have also demonstrated inhibitory potential. To investigate the effect of adipocytes, Han et al. cotransplanted adipocytes and osteoblasts into a mouse model and observed that adipocytes could promote enhanced osteogenesis and reduced osteoclast density as opposed to osteoblasts alone. ⁷⁶ Furthermore, cellular components and secretions have also been investigated. Zheng et al. developed an injectable methylcellulose hydrogel-containing osteocyte lysate. ⁷⁷ The presence of osteocyte lysate was associated with a reduction in TRAP-positive cells within coculture and mouse defect models when compared with controls. It was also associated with significant elevation in new bone formation within the defect models relative to controls. Alternatively, Liu et al. incorporated BMSC-derived extracellular vesicles into a gelatin-based hydrogel to treat periodontitis in rats. ⁷⁸ This system was associated with less TRAP-positive cells and less overall bone loss than a vesicle-free PBS-hydrogel control within the disease model. These findings suggest that future work is needed on the ways through which different cellular factors influence OcLCs in relation to regenerative outcomes. This could enable the isolation and leveraging of specific cellular components for BTE.

Stimulatory soluble factors

Although pharmaceutical-based BTE strategies chiefly exhibit antiosteoclastic properties, there do exist interesting counterexamples (Table 2). For instance, a more complex effect of bisphosphonates on OcLC differentiation has been observed. Li et al. demonstrated this by developing hyaluronic acid-based hydrogels loaded with a bisphosphonate called pamidronic acid. ⁷⁹ The authors’ in vitro studies demonstrated that these hydrogels could support early differentiation of macrophages into preosteoclasts while inhibiting late-stage differentiation into mature osteoclasts as compared with a bisphosphonate-free control. The in vivo studies demonstrated that these hydrogels could enhance bone regeneration compared with both a bisphosphonate-free and a hydrogel-free control. This reveals how bisphosphonates applied in BTE can exert different effects at different stages of OcLC differentiation despite consistent inhibition of osteoclastogenesis. Therefore, an important area for future work will be on the potentially complex ways through which different drugs influence OcLCs within a BTE context.

As discussed in 4.1.1, BMP-2 has shown pro-osteoclastogenic and regenerative properties in multiple BTE studies. Ning et al. developed an injectable composite hydrogel by modifying low-acyl gelatin gum hydrogels with hemicellulose polysaccharide microfibers to treat medication-related osteonecrosis of the jaw (MRONJ). ⁸⁰ The hydrogel was laden with adipose tissue-derived stem cells and BMP-2 and transplanted into MRONJ sockets of rats. These hydrogels stimulated bone formation and osteoclastogenesis relative to those without BMP-2. Interestingly, Brierly et al. evaluated a star-shaped PEG and maleimide functionalized heparin hydrogel (starPEG-heparin) for delivery of BMP-2 in a rat model of MRONJ. ⁸¹ The hydrogel was modified through the functionalization of arginylglycylaspartic acid (RGD) to create starPEG-RGD-heparin hydrogels with BMP-2. The authors observed that hydrogels laden with BMP-2 increased bone formation relative to the absence of BMP-2 and osteoclast number relative to an empty defect. Taken together, these studies show pro-osteoclast and regenerative properties of BMP-2. However, the findings in 4.1.1 demonstrated that the former property can be overridden with additional components like OPG or OP3-4. Therefore, future work ought to uncover the properties and utility of BMP-2 across a greater breadth of BTE contexts.

Multiple groups have also investigated parathyroid hormone (PTH). Yi et al. created a PEG-PCL-PEG (PECE) copolymer hydrogel loaded for continuous PTH (cPTH) or intermittent PTH (iPTH) release and injected it into the midpalatal suture of rats. ⁸² The cPTH hydrogels were associated with increased RANKL expression and inhibited OPG expression when compared with the control and pure maxillary expansion groups. Furthermore, significant increases in bone volume fraction were observed with both cPTH and iPTH release relative to the pure expansion group. Lu et al. also explored local and continuous application of PTH, as well as PTH-related protein (PTHrP). ⁸³ Temperature-sensitive PECE hydrogels carrying PTH and PTHrP were implanted into the maxillary first molars of rats. They found a significant increase in markers of osteoclastogenesis, including osteoclast number and RANKL to OPG ratio, as well as an increase in expression of osteogenic markers such as osteocalcin, RUNX2 and alkaline phosphatase relative to PTH-free and PTHrP-free controls. Alternatively, Hayden et al. developed a culture system with a silk fibroin sponge or film. ⁸⁴ MSCs alone or with osteoclasts derived from THP-1, a specific human leukemia monocytic cell line, were seeded along with tethered PTH or glucose-dependent insulinotropic peptide. Overall, the most notable finding was that incorporating PTH increased calcium deposition and remodeling-induced surface roughness under coculture conditions relative to a green florescent protein control. These findings establish PTH as both stimulatory to OcLCs and effective for bone regeneration. However, more work is needed on the differences between cPTH and iPTH as well as how PTH can be applied to BTE.

Stimulatory environmental factors

As discussed in 4.1.2, ceramics have been extensively investigated in BTE research and frequently are associated with both OcLC stimulation and bone regeneration (Table 3). The most common category would be calcium-based ceramics, which have a particular tendency toward this combination of properties when incorporated in a BTE scaffold. For instance, Jeon et al. investigated the differentiation of osteoblasts and osteoclasts from human-induced pluripotent stem cell-derived MSCs and macrophages on HA-coated PLGA/poly(L-lactic acid) (HA–PLGA/PLLA) scaffolds. ⁸⁵ The group found that the HA-PLGA/PLLA scaffolds with 5.0% HA led to the greatest osteoblast and osteoclast formation in vitro as opposed to 0% or 1% and promoted new bone formation in a murine model. Capitalizing on another calcium-based ceramic, Trojani et al. fabricated a self-hardening Si-hydroxypropylmethylcellulose hydrogel-containing biphasic (40% TCP and 60% HA) calcium phosphate particles. ⁸⁶ It was laden with MSCs and injected into a mouse model. At 4 and 8 weeks, TRAP-positive osteoclastic cells were observed at the injection site attached to the particles with mineralized bone formed in the surrounding area. However, no evidence of bone formation was demonstrated by cell-free and particle-free controls. Considering other material classes, Ji et al. compared 3D scaffolds comprised of gelatin-methacryloyl, PCL, and β-TCP in vitro and within a critical-sized bone defect of rats. ⁸⁷ They observed that the β-TCP scaffold specifically could increase osteoclastogenesis and expression of relevant genes. It was also capable of promoting bone regeneration beyond the control condition. These findings support the notion that calcium-based ceramics are largely stimulatory to OcLCs and conducive to bone regeneration. Further investigations should attend to whether this consistently holds true across various BTE contexts.

Calcium-based ceramics have also been explored in comparison with combined and noncalcium-based ceramics. The work of Borciani et al. exemplifies the former. ⁸⁸ They developed a collagen-based scaffold, hybridized with either HA nanoparticles or strontium-enriched bioactive glasses and evaluated its effect on bone formation in a 2–3-week coculture of osteoblasts and osteoclast precursors. While both scaffolds were observed to promote the development of TRAP-positive cells, the effect was stronger for those containing HA nanoparticles. Both scaffolds could also promote an increase in osteoblast viability over time. In addition, Douglas et al. developed an alkaline phosphatase-loaded GG hydrogel placed in media containing varying ratios of calcium glycerophosphate and magnesium glycerophosphate. ⁸⁹ The resulting mineralized hydrogels’ influence on osteoblast or osteoclast precursors was then investigated. Samples containing proportionally more calcium glycerophosphate led to more osteoclastogenesis, whereas the more magnesium-dosed counterpart promoted more osteoblast activity. However, both calcium- and magnesium-containing samples underwent effective mineralization. Taken together, these results suggest that calcium-based ceramics are an effective means of stimulating OcLCs in a BTE context when compared with alternative ceramics that have been investigated up to the present.

Noncalcium-based ceramics have also been evaluated in association with calcium-based ceramics in the context of OcLC stimulation. For instance, Kowal et al. developed amorphous multiporous scaffold consisting of 70 mol% SiO₂ and 30 mol% CaO laden with osteoblasts and osteoclast precursors. ⁹⁰ The authors observed the promotion of osteoclastogenesis that was absent in controls alongside support of osteoblastogenesis. In conjunction with Panzavolta et al., this indicates that different combination strategies can impact OcLCs in stimulatory or inhibitory directions. ⁶⁷ Lastly, some groups have evaluated noncalcium-based ceramics on their own. Laurenti et al. developed 2D magnesium phosphate nanosheets, designed to be an injectable hydrogel system. In in vivo experiments, it was associated with both enhanced osteoclast and osteoblast number, as well as improved bone defect healing, when compared with a control condition. ⁹¹ These interesting results establish the need for future investigations on OcLC-stimulating noncalcium-based ceramics applied to BTE.

As discussed in 4.1.2, BTE researchers have explored the manipulation of scaffold properties as a BTE strategy through which OcLCs can be modulated. Interestingly, strategies of this nature have also demonstrated stimulatory effects in certain contexts. Exemplifying this, Jones et al. examined different degradable scaffold biomaterials in single or coculture. ⁹² They seeded both osteoblasts and osteoclasts in monoculture or coculture onto scaffolds comprised of vapor-stabilized fibroin, methanol-stabilized fibroin, chitosan, or PLLA. Vapor-stabilized fibroin, methanol-stabilized fibroin, and chitosan were associated with increased osteoclast and osteoblast growth in both monoculture and coculture, whereas PLLA was only conducive to increased osteoblast activity. An interesting avenue for future work is whether this holds true with other synthetic polymers. Alternatively, Wang et al. explored the influence of matrix stiffness on OC activity. ⁹³ The authors observed hydrogel stiffness to be positively correlated with osteoclast formation. They also observed that moderate stiffness was conducive to preosteoclast-dependent osteogenesis and bone regeneration. The efforts of these groups illustrate a need for future investigations to thoroughly explore the influence of mechanical and surface properties on OcLCs in relation to BTE.

Certain nano-scale components have also shown potential in stimulating OcLCs and enhancing bone regeneration. For instance, Croes et al. investigated silver (Ag) nanoparticles as part of a chitosan coating for titanium implants designed to prevent implant infection. ⁹⁴ When incorporated into the tibia of rats, the proinflammatory activity of Ag nanoparticles led to increased osteoclast number and improved peri-implant bone volume relative to a pure chitosan coating. This helps establish nano-scale components as an effective way to stimulate OcLCs in BTE, but more work is needed in characterizing their varying effects.

Stimulatory cellular factors

Myriad investigations have shed light on the potential of cell-based BTE strategies to stimulate OcLCs (Table 4). One area of investigation has been in the use of ObLCs, such as with Zehnder et al., who bioprinted a scaffold with an alginate and gelatin-based hydrogel containing osteoblast and osteoclast progenitors. ⁹⁵ These scaffolds exhibited greater TRAP activity and matrix protein concentration than scaffolds with osteoclast progenitors alone. Alternatively, Chen et al. directly compared an osteoblast-to-osteocyte transition cell line, IDG-SW3, with MSCs in coculture with osteoclast precursors on a 3D collagen hydrogel. ⁹⁶ They found that IDG-SW3 was associated with superior osteoclast activation and a higher quality of matrix deposition in vitro. They also observed an enhanced bone healing when IDG-SW3 was incorporated in vivo. Altogether, the former studies lend support to the potentially OcLC-stimulating and regenerative properties of ObLCs. However, further work is needed on the conditions in which this trend applies across different BTE contexts.

Certain alternative cell types have also demonstrated both stimulatory and regenerative potential. Interested in cancer cells, Quent et al. used tissue-engineered bone constructs with a PEG-based hydrogel containing MDA-MB-231, MDA-MB-231BO, and SUM1315 human breast cancer cells. ⁹⁷ These cells were associated with increased osteoclast number in comparison with the scaffold alone in in vivo studies. In addition, new bone formation occurred around the constructs, indicating compatibility between these cells and bone regeneration. Interestingly, Nagata et al. performed a clinical trial investigating the application of cultured autogenous periosteal cells (CAPCs) in promoting alveolar bone regeneration. ⁹⁸ These CAPCs were implanted after being mixed with autogeneous bone and platelet-rich plasma across 33 sites in 25 cases. This treatment was associated with effective osteoblast and osteoclast recruitment, as seen by elevations in alkaline phosphatase and TRAP activity within regenerated bone tissue with respect to CAPC-free controls. While this clinical trial stands as one of the only of its kind, these results demonstrate a potentially promising future for OcLC-stimulating cell-based constructs. Overall, these studies reveal an intriguing influence of certain alternative cell types on OcLCs and bone regeneration that warrants more thorough future investigation.