Bone matrix mainly consists of the inorganic component, hydroxyapatite, and the organic component, type I collagen, which is a calcified intercellular matrix (8). These components constitute the infrastructure of the mechanical properties of bone, e.g., tensile and compressive strength. Four primary cell populations present in bone tissue are osteoprogenitor cells, osteoblasts, osteoclasts, and osteocytes (9). Osteocytes are predominantly located within the bone matrix, while the other three cell types are found at the edge of bone tissue. The maintenance of bone homeostasis hinges on the dynamic balance between the osteoclast and osteoblast.

Osteoblasts are mainly involved in the development of new bone and undergo three differentiation stages: osteoprogenitor cells, preosteoblasts, and osteoblasts. Osteoblasts are derived from MSCs through multiple differentiation pathways. Initially, in osteoblast cell lines, MSCs differentiate into osteoprogenitor cells (10). The expression of Sox family transcription factors marks the primary differentiation of osteoblasts. Sox4 and Sox11 (SOXC group) promote the survival of osteoprogenitor cells (11), while Sox9 indicates differentiation toward chondrocytes (12). Osteoblasts could transform into osteocytes, which interact with nerves, blood vessels, and tissue fluid to form osteons that provide structural support for the skeleton.

In addition, bone morphogenetic proteins (BMPs) also play a critical role in bone generation and repair (13), attracting the aggregation of preosteoblasts at the injured site and differentiating them into osteoblasts (14). Recombinant human bone morphogenetic protein-2 (rhBMP-2) was approved by the FDA in 2002 for use in anterior lumbar interbody fusion. However, complications have been identified, such as excessive osteogenesis, osteolysis, inflammation, edema, and carcinogenicity.

Osteoclasts originate from hematopoietic stem cells, and their function is mainly to absorb and resorb bone. Hematopoietic stem cells can differentiate into osteoblasts, macrophages, dendritic cells, and other cell types (15). Macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), which can be produced by stromal cells, osteoblasts, and immune system cells, are essential in the differentiation and survival of osteoclasts (16). Interestingly, they are highly expressed in osteoblasts (17). M-CSF binds to the CSF-1 receptor to activate downstream signaling and further upregulate RANK expression, thereby promoting the differentiation of hematopoietic stem cells into osteoblast precursor cells (18). When activated by RANKL-RANK signaling, osteoclast precursor cells differentiate, fuse, and interact with osteoblasts to become mature osteoclasts (19). Osteoprotegerin (OPG) is also involved in this process. OPG, produced mainly by osteoblasts, is a soluble RANKL decoy receptor that inhibits osteoclast formation and bone resorption by preventing RANKL-RANK receptor interactions (20). M-CSF can stimulate cell survival signaling through activation of thymoma virus proto-oncogene 1 (commonly known as AKT) via phosphatidylinositol 3 kinase (PI3K), but it most notably activates extracellular signal-regulated kinase (ERK) via growth factor receptor binding protein 2 [Grb-2; (21)]. All these factors can indirectly induce osteoblast precursor cells to differentiate into mature osteoblasts (Figure 1).

The growth and development of human bone involve a continuous process of bone remodeling. In this process, osteoclasts absorb old bone matrix, and osteoblasts deposit sedimentary new bone matrix (8). However, bone regeneration after damage caused by trauma, tumors, or inflammation is a distinct regenerative process. This process commences with the onset of inflammation and the upregulation of proinflammatory factors at the site of damage, including tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), interleukin-6 (IL-6), and interleukin-11 (IL-11) among others (22). Subsequently, neutrophils and macrophages accumulate at injury site. The bone remodeling process occurs in four stages (19): recruitment and activation of osteoclasts at the site of injury; resorption of broken bone matrix by mature osteoclasts; differentiation of osteoprogenitor cells into mature osteoblasts; and aggregation of osteoblasts to form new bone matrix and mineralization. These stages are distinct and overlap with each other.

The mechanisms underlying bone regeneration have been extensively investigated, leading to the identification of various signaling pathways involved in this process (23). These pathways, such as the Hedgehog signaling (Hh) pathway, Notch signaling pathway, WNT signaling pathway, BMP/TGF-β and MAPK signaling pathway, IGF signaling pathway, and other signaling pathways (FGF signaling pathway, PI3K/Akt/mTOR signaling pathway, et al.), interact with each other to co-regulate bone regeneration (24) (Figures 2–5).

Bone development and regeneration involve a complex interplay of various signaling pathways that coordinate and crosstalk with each other (49). Despite the distinct roles of these pathways, they function cooperatively to ensure proper bone formation and repair. The interaction between the TGF-β/BMP pathway and the Wnt pathway is critical for early development and tissue stabilization (58). TGF-β can prevent hyperphosphate-induced osteogenesis by inhibiting the Wnt/β-catenin pathway and reducing nuclear translocation of Smad 1/5/8 in the Smad-dependent BMP signaling pathway (59). Furthermore, Wnt3a inhibits Smad1 phosphorylation via GSK3 and stabilizes Smad1 (60). At Ser-204 and Ser-208 (61), GSK3 mediates Smad3 phosphorylation and inhibits its activity. Axin promotes GSK3β-mediated Smad3 phosphorylation at Thr66, leading to Smad3 ubiquitination and degradation (62). TGF-β regulates Hes-1 transcription in a Notch-dependent manner. The intracellular domain of Notch1 (NICD) can interact with Smad3, enhancing cascade signaling (63).

Although numerous pathways not listed here are also involved in bone regeneration and development, the mechanisms underlying skeletal development and repair are primarily a result of cross-talk between these pathways.

DPSCs have emerged as a prospective cell source for stem cell-based therapies that can be readily available from third molars, deciduous teeth, or permanent teeth (69). Third molars are usually discarded as medical waste, and two sets of teeth germinate during a person’s lifetime, indicating that DPSCs are abundant and easily accessible. DPSCs have MSC-like properties, such as multidirectional differentiation and the ability to self-renew. Compared with other sources of MSCs or progenitor cells, DPSCs are not only easy to obtain but also have low immunogenicity and avoid ethical concerns. More importantly, DPSCs can be collected without injury to the donor or invasive surgical procedures (70).

Because of their origin in the cranial neural crest lineage, DPSCs have significant neural differentiation potential, making them suitable in treatment of neurological problems (71). However, their advantages in bone regeneration are also unquestionable. DPSCs exhibit higher fibroblast colony-forming units and proliferation rates, similar gene expression profiles of mineralization-related genes, and differential osteogenic, paracrine, and immunomodulatory capacity as compared to BMMSCs (72). The osteoblasts in the craniofacial region are also derived from neural crest cells, which are derived from neural ectoderm. During the embryonic period, the neural ectoderm develops into the tissues of the craniofacial region, including the facial bones, the skull, and the dentin of the teeth (23). Therefore, DPSCs have great osteogenic potential (73).

In the past few years, the majority of scholars have shifted their focus to inducing bone regeneration through the transplantation of BMMSCs, adipose-derived mesenchymal stem cells (ADSCs), and umbilical cord-derived mesenchymal stem cells (UCMSCs). Firstly, bone marrow mesenchymal stem cells are heterogenous cell populations located in the medullary stroma of bone marrow, originating from the early development of the mesoderm and outer layer. They are the earliest-discovered and extracted type of mesenchymal stem cells. BMMSCs possess a strong ability to differentiate into bone cells and can be used for repairing bone defects (74). By implanting BMMSCs into the defective area, it is possible to promote bone tissue growth and reconstruction (75). However, due to their low proliferation capacity, high collection risks, and the painful collection process, researchers have been searching for alternative sources of mesenchymal stem cells (76). Subsequently, adipose stem cells and umbilical cord mesenchymal stem cells have been applied to treat bone defects. ADSCs, compared to other stem cells, exhibit excellent proliferation and differentiation potential (77), capable of promoting tissue regeneration and functional recovery. UCMSCs possess strong immune regulation and anti-inflammatory abilities (78), are able to alleviate inflammatory reactions and promote tissue recovery, but have a relatively weak osteogenic capability and are associated with ethical controversies (79).

Like other types of stem cells, DPSCs exert their therapeutic effects primarily through paracrine signaling. Specifically, DPSCs secrete a wide range of bioactive molecules, including regulatory factors, growth factors, cytokines, and signal peptides (80). DPSCs also release secretory proteomes, including exosomes, microvesicles, apoptotic vesicles, and other extracellular factors (81), which act in the body to provide a suitable environment for immune regulation and anti-apoptosis, maintaining overall body homeostasis. Importantly, DPSCs exhibit low immunogenicity (82), and the expression of histocompatibility complex class II antigen does not exist on DPSCs. These unique properties have led to the development of various therapeutic applications for DPSCs in treating neurological, circulatory, diabetic, liver, eye, immune, and oral diseases (83).

The regenerative potential of dental pulp stem cells (DPSCs) in the field of craniofacial bone repair is not to be underestimated. Gaus et al. conducted a study using data from the Gene Expression Omnibus database, which revealed 16 overlapping differentially expressed miRNAs and shared regulators associated with bone differentiation between DPSCs and BMMSCs, suggesting a common genetic and epigenetic mechanism for bone differentiation in both cell types (84). An in vitro study has demonstrated that DPSCs exhibit superior osteogenic potential compared to other MSCs, including BMMSCs, gingival mesenchymal stem cells (GMSCs), and adipose-derived stem cells (ADSCs), as evidenced by various assays such as fluorescence-activated cell sorting, flow cytometry, quantitative polymerase chain reaction for osteogenic gene expression, alizarin red staining, and micro-computed tomography analysis (85). However, other conflicting results have been reported (72, 86–94), which may be attributed to differences in MSCs donors and isolation techniques. Table 1 summarizes the inconsistent findings on the osteogenic potential of DPSCs. Nevertheless, DPSCs show advantages in terms of proliferation rates, cell utilization, and cell numbers compared to BMMSCs (90, 92). These advantages are reflected in Table 2. DPSCs also exhibit remarkable immunomodulatory, paracrine, anti-apoptotic, and angiogenic properties, which contribute to bone differentiation and reduce inflammation (91, 96, 98, 101). Anderson et al. showed that DPSCs reduce inflammation and induce M2 polarization in bone marrow cells (102). The same conclusion was drawn in vivo. In a rabbit cranial defect model, inoculation of BMSC or DPSC in a Bio-Oss stent and implantation into a 6-mm cranial defect promoted bone regeneration and improved osteogenesis-related protein expression at the defect site, and the bone regeneration efficacy of the two cells was shown to be compatible (94). In a temporomandibular joint arthritis rat model, local injection of DPSCs was found to alleviate inflammation and pain in the joint cavity, promote bone regeneration, and inhibit the STAT1 signaling pathway (103).

The ability of DPSCs to promote bone regeneration is mediated by various mechanisms. Ferutinin, a phytoestrogen, promotes osteogenic differentiation of the Wnt/β-catenin signaling pathway by activating H3K9 acetylation and H3K4 trimethylation in the promoter regions of Wnt3a and DVL3 (104). Similarly, adenosine A1 receptors have been shown to promote DPSCs osteogenesis through the Wnt/Dvl pathway, as evidenced by increased expression of the osteogenic markers RUNX-2 and ALP (105). AR-A014418, a glycogen synthase kinase 3β (GSK3β) inhibitor, promotes not only the proliferation and migration of DPSCs but also their osteogenic differentiation, which is achieved through the activation of the β-catenin/PI3K/Akt signaling pathway (106). DPSCs also inhibit osteoclasts by inactivating the AKT pathway through the secretion of OPG, as demonstrated by Kanji et al. (107). Melatonin affected the osteogenic differentiation ability of DPSCs through COX-2/NF-κB and p38/ERK MAPK signaling pathways, which was also verified in the rabbit calvarial defect model (108). In addition, some drugs, enzymes, hormones, or trace elements can induce DPSCs to express osteogenic-related genes through the BMP4/Smad pathway, the Erk1/2 pathway, the p38 MAPK pathway, and other pathways to promote bone regeneration and repair (109, 110). Notably, the formation and connectivity of blood vessels are crucial for osteogenesis (111), and DPSCs have been shown to possess a higher angiogenic potential than other MSCs (112).

MSCs are known to secrete a variety of substances, including numerous proteins, peptides, RNA, and lipid mediators, as well as an abundance of extracellular vesicles (EVs). Mesenchymal Stem Cell Conditioned Medium (MSC-CM) (121) is derived from the supernatant of cultured MSCs and is a mixture of hundreds to thousands of different enzymes, growth factors, cytokines, and proteins that can be concentrated, frozen, or even lyophilized without loss of activity. MSC-CM can be collected in a simple and efficient manner. Cells are inoculated in a culture dish and cultured with serum-containing medium until the density is approximately 70%, after which serum-free medium is added, and the culture continues for 48 h. The supernatant is then collected and filtered with a sterile 0.22 μm filter to remove cell debris and bacterial microorganisms. The final supernatant is referred to as conditioned medium for MSCs and can be stored at −80°C until use or further concentrated and stored at ultra-low temperatures (122).

MSC-CM from different sources has been identified to have diverse and beneficial effects on the receptor, including anti-inflammatory, immunomodulatory, and angiogenic effects (123). Huang et al. used periodontal membrane-derived stem cell-conditioned medium, concentrated 20-fold, to culture chondrocytes, synovial cells, and meniscus cells isolated from IL-1β-treated porcine knees. The results validated increased expression of anti-inflammatory factors as well as decreased expression of inflammatory factors, confirming the anti-inflammatory effect of MSC-CM (124). Gharaei et al. analyzed multiple pro- and anti-angiogenic factors in DPSC-conditioned medium (DPSC-CM), cultured with human umbilical vein endothelial cells (HUVEC). They reported that these factors affected cellular migration and proliferation, stimulated tubulogenesis and promoted angiogenesis such as the number of nodes, meshes, and total tubular length (125). Proteomic analysis has uncovered a total of 1,533 proteins in CM derived from ADSCs, BMMSCs, and DPSCs, which have regenerative potential in areas such as cell migration, angiogenesis, inflammatory response, ossification, and organ survival. Moreover, the expression of multiple cytokines associated with odontoblast differentiation and anti-inflammatory cytokines was significantly higher in DPSC-CM (126). Paschalidis et al. found that DPSC-CM enhanced the viability, migration, and mineralization potential of DPSCs and even counteracted TEGDMA-induced cytotoxicity (127). Through in vivo and in vitro studies, Fujio et al. showed that anti-inflammatory, angiogenesis, and osteogenic-related factors were more highly expressed in DPSC-CM under hypoxia and promoted osteogenesis and accelerated bone healing in mouse tibia (128). DPSC-CM also increases mineralization potential through TGF-β1 expression, thereby triggering new bone formation and improving osteoblast and chondrogenic markers (128). New blood vessels are particularly important in the bone regeneration process, allowing better reconnection of new bone to its own bone. Ishizaka et al. showed that in vivo DPSC-CM exhibited higher immunomodulatory capacity as well as higher angiogenic and anti-apoptotic capacity in vivo compared to BMMSCs-CM (96).

All mammalian cell types studied so far, including neuronal cells, endothelial cells, MSCs, and epithelial cells, have been found to release EVs. EVs can be found in all kinds of body fluids like saliva, synovial fluid, urine, and blood. While EVs were previously considered to be cellular waste, recent research has shown that they have a major role in regulating cellular signaling pathways in target cells, including tumor cell growth, cell migration, cell communication, and angiogenesis (129).

EVs are small membranous vesicles that are released into the extracellular matrix by cells via the plasma membrane during the budding process (117). EVs contain a variety of substances, including phosphatidylserine, cytoplasmic proteins, mRNA, miRNA, DNA, and other molecules. Exosomes (Exos) are one group of the smallest and most extensively studied MSC-derived EVs. The genesis of Exos occurs through the endocytic exocytosis pathway, with early endosomes maturing, extending to late endosomes, and budding inward at the late endosomal membrane of multivesicular bodies. After the fusion of these multivesicular bodies with the cell membranes, MSC-derived Exos are released into the extracellular environment. These Exos carry important signaling molecules and exert biological effects in target cells (67, 130).

Various methods are available for the isolation of EVs and Exos, including ultracentrifugation, density gradient centrifugation, exosome kits, ultrafiltration, immunoprecipitation, and acoustic nanofilters (131). Ultracentrifugation is the gold standard method for Exos extraction, which involves multiple rounds of centrifugation at varying speeds to collect Exos from cell culture supernatants (132, 133). Density gradient centrifugation, on the other hand, separates Exos with less contamination but is more complex and time-consuming (134). Recently, commercial kits based on polymer coprecipitation have been developed for Exos extraction, such as ExoQuick, which is simple to use and has a high yield but may contain impurities (135). While each method has its advantages and disadvantages, ultracentrifugation is considered to be the most reliable and efficient technique for Exos extraction. However, this method can be expensive, time-consuming, and require large sample volumes. In contrast, commercial kits are simpler and faster but may have a higher level of impurities. Density gradient centrifugation offers higher purity, but the preparation is more complicated and time-consuming, and Exos cannot be completely separated from proteins. Overall, the choice of isolation method should depend on the specific research needs and resources available.

Exos have emerged as promising candidates for bone defect regeneration (136, 137). Studies have shown that Exos can bind to scaffolds or growth factors and modulate both osteoblast and osteoclast functions (138). Exos have also been found to induce osteogenic differentiation and bone regeneration by increasing osteoblast differentiation-related miRNA expression and inhibiting Axin1-activated Wnt signaling (139). High-throughput miRNA sequencing has revealed that Exos secrete 41 miRNAs that are differentially expressed after osteogenesis induction. These miRNAs have been implicated in bone differentiation and development, including osteoclast differentiation, the PI3K-AKT signaling pathway, the MAPK signaling pathway, and the mTOR signaling pathway. Among these miRNAs, hsa-mir-328-3p and hsa-mir-2110 have been identified as potentially the most important osteogenic regulatory miRNAs in Exos (140). In addition, Wei et al. prepared exosome-treated titanium nanotubes to enhance the osteogenic potential of BMP2 via natural nanocarriers (141).

Moreover, exosomes from human exfoliated deciduous teeth (SHED-Exos) have been shown to stimulate BMMSCs to express more osteogenic-related genes, such as Runx2 and ALP. Surprisingly, they also down-regulate lipopolysaccharide (LPS)-induced expression of inflammation-related factors in BMMSCs.

Although less research has been conducted on the use of dental pulp stem cell-derived exosomes (DPSC-Exos) in bone regeneration, their advantages are evident. DPSC-Exos have been shown to regulate target cells by translocating mRNA, miRNA, proteins, and other molecules to receptor cells. Hu et al. analyzed the microRNA profile of DPSC-Exos and verified that miR-27a-5p of DPSC-Exos promotes odontogenic differentiation of DPSCs by downregulating LTBP1 (one of the suppressor molecules of TGFβ1 signaling) to regulate the TGFβ1/Smads signaling pathway and thus express more osteogenic-related genes (142). DPSC-Exos have also been proven to enhance the proliferation and migration of haplotype homo dental pulp cells and mouse osteoblasts, inhibit the formation of mouse osteoclasts, and effectively reduce bone loss caused by periodontitis (143). Combining DPSC-Exos and DPSCs with β-tricalcium phosphate, hydroxyapatite, or collagen in a rat skull defect model has been found to accelerate bone regeneration and promote more extensive angiogenesis at the defect site. Notably, DPSC-Exos and DPSCs were found to have almost identical effects at the site of bone defects (144). Although DPSC-Exos has been shown to have bone tissue repair effects, the mechanisms behind these effects are not fully understood.

Cell lysate, a product of cell lysis, is a promising source of bioactive compounds for regenerative medicine and tissue engineering applications. Although the specific components of the cell lysate remain uncertain, it is known to contain several soluble nutrients, including growth factors, EVs, Exos, and other proteins. To obtain the cell lysate, cells are first cultured and then lysed using trypsin digestion, followed by centrifugation and resuspension in ultra-pure water, and finally subjected to ultrasound or freeze–thaw technology. At this stage, cell releases various proteins and soluble nutrients, including growth factors like EGF and IGF. It can be used in the same way as MSCs, CMs, EVs, Exos, etc. to treat diseases.

While there is limited literature on the utilization of cell lysate for treating bone defects, several studies have demonstrated its anti-inflammatory and regenerative properties in other applications. For example, Jiao et al. were surprised to find that MSC-conditioned medium (MSC-CM) or MSC lysate (MSC Ly) can substantially enhance IL-10 secretion by peripheral blood mononuclear cells (PBMCs) in vitro. Simultaneously, it can significantly increase serum IL-10 levels in two animal models and reproduce the effects of an MSC graft in vivo (145). Ward et al. used the classical hind-paw edema model to simulate temporomandibular joint osteoarthritis, after treatment with human umbilical perivascular mesenchymal cells and their lysates, there were significantly lower concentrations of myeloperoxidase and TNF-α at 48 h. Treated osteoarthritis demonstrated lower concentrations of leukocytes in the synovium compared to controls and histologic evidence in the peri-articular tissue of reduced inflammation (146). Similarly, lysates of olfactory mucosa tissue-derived mesenchymal stem cells (OM-MSCs), when cultured with LPS-stimulated normal human liver cells (LO-2), inhibited the inflammatory process and promoted the proliferation rate of LO-2. Similarly, in a mouse model of LPS-induced acute liver injury, OM-MSCs lysate treatment attenuated inflammation and reduced liver enzyme release, thus alleviating liver injury (147). Khubutiya et al. reported that a mixture of BMMSC-conditioned medium (BMMSC-CM) and MSCs lysate significantly enhanced liver regeneration and reduced injury in a mouse model of acetaminophen-induced acute liver injury (148). Moreover, the administration of filtrated adipose tissue-derived mesenchymal stem cell lysate for three consecutive days mitigated inflammation and inhibited cell apoptosis in a mouse model of acute colitis, leading to improved survival rates, reduced weight loss, and clinical signs (149). Erectile dysfunction (ED) remains a major complication after radical prostatectomy. Albersen et al. found that penile injection of both ADSC and ADSC-derived lysate can improve recovery of erectile function in a rat model of neurogenic erectile dysfunction (150). Our research team has found that the lysates of DPSCs can be used for anti-photoaging treatment, and ZIF-8, as a safe and effective protein delivery system, can improve the skin bioavailability of DPSC lysates, demonstrating significantly enhanced cell uptake and skin retention ability (151). The cell lysate extraction process is rapid, simple, and produces a diverse range of precipitated components with excellent efficacy, thus holding great potential for future applications in regenerative medicine and tissue engineering.

Recent experimental and clinical studies have shown that the use of the MSC secretome is a highly successful therapeutic strategy. MSC-CM and MSC-derived EVs have demonstrated similar therapeutic efficacy to MSC transplantation in bone and cartilage defects (152). The replacement of cell therapy with cell-free therapy eliminates the undesirable side effects associated with live cell transplantation, including immune rejection, emboli formation, tumorigenicity, arrhythmias, calcified ossification, and disease transmission. The secretome can be stored at ultra-low temperatures, such as −80°C, for prolonged periods of time. Cryopreservation or freeze-drying does not compromise the effectiveness of the cell secretome. Whereas cell cryopreservation necessitates the use of potentially toxic cryopreservation agents and harsh cryopreservation temperatures. In emergency situations, the secretome can be rapidly thawed for immediate use. Lastly, following clinical translation, the cell secretome can be standardized in terms of dose and potency, akin to a clinical drug (153).

Despite all the benefits of cell-free therapy, there are some limitations to its application. For instance, there are currently no standardized protocols for producing large quantities of EVs or extracellular vesicles. Furthermore, soluble factors secreted by MSC have not been fully elucidated. And it is unclear whether the use of CM, EVs, or cell lysate is safe for treating various diseases. Most significantly, the legal regulations surrounding cell-free therapy have not yet been fully established, given the rapid evolution of this field. Cell-free therapy remains in its nascent stages, and additional research is required to fully understand its potential as a therapeutic strategy (154).