Utilizing key developmental cues and refining their orchestrating role in degeneration represents a promising strategy for understanding and treating intervertebral disc (IVD) degeneration, a major cause of chronic lower back pain. Here, we focus on notochordal cells (NCs), which originate from the embryonic notochord and reside in the developing nucleus pulposus. These distinctly vacuolated cells exhibit robust regenerative effects and hold promise for new therapeutic approaches. Dogs, like humans, suffer from the consequences of IVD degeneration. As the IVD matures and degenerates, NCs are replaced by smaller non-vacuolated NP cells (NPCs). The dog was employed as a model to capture, at the single-cell level, the heterogeneity of resident cells by studying the nucleus pulposus tissue at three stages (i.e., juvenile, young adult and degenerate adult). Here, we integrated transcriptomic data with repressive histone H3 lysine 27 trimethylation (H3K27me3) profiles at the single-cell level to assess changes in chromatin states and gene expression across this IVD degeneration-associated cell phenotypic transition. H3K27me3 enrichment on key genes involved in IVD development and homeostasis, such as Brachyury (TBXT), aligns with the observed attenuation during ageing and degeneration seen in both dog and human IVDs. This study further demonstrates that eliminating repressive histone marks, together with CRISPR-mediated gene transactivation, enhances TBXT gene expression in human NPCs derived from degenerated aged discs. Our findings underscore how extensive insights gained through single-cell omics can lead to the identification of crucial cellular cues that may enable degenerate NPCs to regain a healthier phenotype.
Ossification of the posterior longitudinal ligament (OPLL) is a chronic degenerative spinal condition in which bone gradually forms within the ligament, compressing the spinal cord and causing progressive neurological decline. While genetic predisposition and metabolic disturbances are well-recognized contributors, growing evidence points to macrophages as central players in shaping the tissue environment that drives pathological bone formation. Macrophages are remarkably plastic cells whose phenotypes and functions are continuously molded by cues from their local tissue environment. While pro-inflammatory macrophages sustain chronic low-grade inflammation through cytokine release and matrix remodeling, anti-inflammatory macrophages paradoxically amplify disease progression by driving fibrotic remodeling, excessive matrix accumulation, and aberrant vascularization, collectively fostering a hypoxic, ossification-prone niche within the ligament. Supporting this notion, similar macrophage-mediated mechanisms have been described in heterotopic ossification, though OPLL represents a distinct pathological process. This review synthesizes current evidence on how macrophages shape the OPLL microenvironment, draws mechanistic connections between macrophage activity and disease progression, and explores whether targeting these cells could open new therapeutic avenues.
Osteoarthritis (OA) is a widespread degenerative joint disorder manifesting as chronic pain, functional impairment, and progressive disability, imposing a substantial burden on global public health. Despite extensive research, the precise molecular pathways underlying OA pathogenesis remain incompletely elucidated. Our data indicated that human OA cartilage and cartilaginous tissues from aged mice exhibited markedly elevated levels of extracellular signal-related kinase 5 (ERK5) enzyme activity. Using Col2a1-CreERT2/Erk5flox/flox mice, we found that Erk5 deletion in cartilage inhibited the progression of post-traumatic and aging OA mouse while constitutive activation of ERK5 significantly accelerated OA development via intra-articular injection of adeno-associated virus model. Mechanistically, ERK5 interacted with the PYD domain of NLRP3 and mediated NLRP3 phosphorylation at serine 198, which facilitated mature inflammasome assembly, triggered pyroptosis and subsequently exacerbated OA progression. Furthermore, we identified a small compound, Oroxylin A, which attenuated ERK5 activation and effectively ameliorated the development of post-traumatic and aging-induced OA in mice. Taken together, our studies demonstrate that ERK5 is a key regulator effecting chondrocytes inflammation and pyroptosis through activating NLRP3 inflammasome and ERK5 is a potential therapeutic target for OA treatment.
Mechanical stimulation is fundamental for anabolism in various tissues, including bone. Importantly, mechanical stimulation has been shown to induce reprogramming of cell metabolism, likely to adjust it to increased anabolism. The signal transduction within this mechano-metabolic axis is incompletely understood. Aiming to delineate such signal transduction, we exposed osteoprogenitors to fluid shear stress (FSS) and assessed cell signaling and bioenergetics. The BMP/Smad pathway is known to be activated by mechanical stress, and we indeed detected its activation by FSS. We previously reported that BMP downregulates cyclophilin D (CypD), an opener of the mitochondrial permeability transition pore (MPTP). Downregulation of CypD/MPTP improves mitochondrial inner membrane integrity and therefore oxidative function. We found that in FSS-stimulated cells, CypD was indeed downregulated and mitochondria were activated in a BMP-dependent manner. Meanwhile, the osteogenic effect of FSS was dependent on CypD downregulation and mitochondrial responses. To confirm our in vitro results in vivo, we optimized a novel model of tooth extraction-mediated unloading of craniofacial bones in mice. Such unloading led to bone loss and upregulation of CypD in the affected bone. Osteoblast-specific deletion of CypD protected against unloading-mediated bone loss, while CypD re-expression in these mice restored bone loss. In sum, we here present new evidence that the mechano-metabolic axis in osteogenic cells involves BMP/Smad-mediated downregulation of CypD and CypD-dependent mitochondrial responses. Such regulation is important for the osteoanabolic effect of mechanical stimulation. Our data also suggest that targeting CypD can be an effective strategy to prevent bone loss caused by unloading due to immobility, space flight, or tooth extraction.
Interoception is the process by which the nervous system senses, integrates, and interprets signals arising from within the body, thereby enabling the brain to monitor and regulate physiological functions. Often described as a "sixth sense", interoception links internal physiological states to brain-mediated homeostatic control, whereas sight, smell, hearing, taste, and touch primarily convey information from the external environment. Organoids are three-dimensional tissue models derived from stem or progenitor cells that reproduce selected structural and functional features of native organs. Because organoids are increasingly used in disease modeling, drug testing, and regenerative research, their integration with neural, endocrine, immune, and vascular components offers an opportunity to investigate brain-body communication. Conversely, organoid-based systems may provide experimentally tractable models for interoception. This review defines interoception and organoid technologies, examines brain-centered communication with skeletal and intestinal systems, and discusses how brain, bone, and intestinal organoids and assembloids could advance interoception research. In particular, organoid-based models may enable mechanistic investigation of neuroendocrine pathways, including the hypothalamic-pituitary-adrenal (HPA) axis, and thereby provide new insights into physiological regulation and disease progression.
Biomolecular condensates are membraneless assemblies that concentrate proteins, nucleic acids, and other biomolecules into dynamic cellular compartments. Liquid-liquid phase separation (LLPS) is one important route by which such condensates form, particularly when multivalent interactions generate liquid-like, reversible assemblies. However, not every condensate or disease-associated assembly should be explained solely by LLPS. In the musculoskeletal system, condensates have been linked to transcription, signal transduction, RNA metabolism, stress responses, tissue development, homeostasis, and mechanoadaptation. Genetic mutations, altered post-translational modifications, and environmental stress can disturb these assemblies, but the evidence does not always establish whether condensates are causal drivers of disease or downstream responses to injury. This review examines how biomolecular condensates and LLPS-related mechanisms have been implicated in osteoporosis, osteoarthritis, skeletal muscle atrophy, bone and soft tissue sarcomas, and neuromusculoskeletal diseases. We focus on the strength of the available evidence, distinguish correlative observations from causal mechanisms where possible, and discuss how condensates may connect non-coding genetic variants, mechanical cues, metabolic signals, and disease phenotypes. We also assess the translational potential and limitations of condensate-based biomarkers, therapies that target pathological condensates, and phase-separation-inspired delivery systems. A central message is that biomolecular condensates offer a useful framework for musculoskeletal biology, but clinical translation will require disease-specific targets, human-relevant models, selective delivery, and rigorous tests of causality.
A rate-limiting step in the prevention and early intervention of osteoporosis is identifying its asymptomatic onset. Accumulating evidence shows that sleep disorders are associated with an increased risk of osteoporosis. Given their early detectable and modifiable nature, integrating sleep disorder management into osteoporosis prevention and care pathways offers a novel approach for enhancing skeletal health. This expert consensus represents a collaborative effort by specialists in sleep medicine and orthopedics from across the world, integrating epidemiological, mechanistic, and interventional evidence to provide general guidance for prevention and clinical practice, and to foster multidisciplinary collaboration in the management of sleep disorders and osteoporosis.
Interoception is a core process through which the body perceives its internal state and regulates physiological homeostasis via bidirectional communication between the central and peripheral nervous system. Skeletal interoception is a specific circuitry for the brain control of the weight-bearing system, particularly responsible for sensing bone-derived internal signals to maintain skeletal homeostasis in response to mechanical loading. Recent studies uncovered that prostaglandin E2 (PGE2) plays a crucial role in skeletal interoception, and is therefore involved in major skeletal disorders and pain conditions such as low back pain, osteoarthritis and particularly ankle osteoarthritis (AOA). Ankle pain is clinically common, with a prevalence of 9%-15% among adults, severely impairing work productivity and quality of life. This article reviews the progress of skeletal interoception in skeletal pathogenesis and pain, with AOA as an example. Specifically, it discusses PGE2 and skeletal interoception in relation to pain and inflammation. We also attempted to interpret non-steroidal anti-inflammatory drugs (NSAIDs), surgical interventions and Traditional Chinese Medicine (TCM) therapies, especially acupuncture and electroacupuncture, in the therapy of pain and osteoarthritis from the viewpoint of skeletal interoception. Interoception is an emerging science in understanding how the brain regulates peripheral organs. Skeletal interoception mediated by PGE2 provides an opportunity to understand the potential of NSAIDs and acupuncture in regulating interoception for the treatment of skeletal disorders including ankle pain.
Exhaustion of skeletal stem and progenitor cells (SSPCs) drives age-related delays in fracture repair, yet the upstream regulators of SSPC maintenance are unclear. We identify that core-binding factor β (Cbfβ) in bone marrow Adipoq+ cells (BMACs) is essential for maintaining SSPC number and function. Cbfβ deletion in BMACs (CKO) leads to SSPC depletion, including periosteal populations, and impairs bicortical fracture healing in mice. Multi-omics (RNA-seq, CUT&Tag-seq, and ATAC-seq) reveal that Cbfβ preserves chromatin accessibility at DNA repair loci, maintaining genomic stability, preventing BMAC senescence, and mitigating the senescence-associated secretory phenotype (SASP). Senolytic therapy alleviates BMAC senescence, restores SSPC populations, and improves bone repair in CKO mice. In both humans and mice, Cbfβ expression declines with aging, accompanied by increased BMAC senescence. AAV-mediated Cbfβ overexpression restores aging-related bone repair and SSPC decline. These findings reveal a novel mechanism in which Cbfβ in BMACs regulates SSPC maintenance via a senescence/SASP axis, offering a potential therapeutic strategy for age-related bone repair deficits.
Maintaining postnatal bone growth is crucial for humans to reach their final height. To determine transcriptional networks coordinating this process, we applied spatially resolved transcriptomics to growth plate biopsies obtained from healthy adolescents who underwent epiphysiodesis surgery for idiopathic tall stature. Spatial profiling revealed new markers for each zone of the human growth plate and identified genes associated with poorly understood growth disorders, including the novel hypertrophic zone marker SGMS2. We elaborated on this finding and established that Sgms2 is present in growth plate-derived matrix vesicles, and its activity facilitates mineralization - a process impaired in patients with SGMS2 mutations. By exploring the low transcriptional activity of resting zone chondrocytes, we found that a subset of these cells exists in a functionally quiescent state in vivo, as determined by their predominantly nuclear mRNA, abundant heterochromatin, and ability to exit the G0 phase under specific conditions - features shared with skeletal stem cells in mouse growth plates. Additionally, we identified distinct sub-populations of human resting zone chondrocytes; an exploration of their hierarchy determined that CHRDL2 and/or SFRP5-positive sub-populations were among the least quiescent resting zone cells. In summary, we generated a comprehensive map of gene expression within the human growth plate, revealing novel zone-specific markers, new primary growth disorders, candidate pharmacological targets, and sub-populations of resting zone chondrocytes with features of quiescent stem cells. These results contribute to a better understanding of the cellular and molecular mechanisms governing human height and can facilitate improved diagnosis and treatment strategies for patients with skeletal growth disorders.
Inflammation causes bone loss by dysregulating the differentiation and functions of osteoclasts, osteoblasts, and osteocytes. This process can be modeled by the expression of constitutively activated IKK2 (IKK2ca), a strategy that we leveraged to investigate the mechanisms through which inflammation negatively affects cells of the osteoblast/osteocyte lineage. We found that mice expressing IKK2ca in osteoblasts exhibit significant bone loss. Mechanistically, IKK2ca downregulates the expression of osteoblast genes while inducing the differentiation of bone-forming osteoblasts into catabolic osteocytes like cells, expressing high levels of Podoplanin, Fgf23, Dkk1 and Sclerostin. We term these atypical inflammatory osteocyte-like cells (aiOCy-L cells) as they highly express inflammatory and senescence markers and promote osteoclast differentiation as well. Additional data show that inflammation induces abnormal differentiation of OB into aiOCy-L cells through upregulating mTOR and glycolysis. In summary, we uncovered a mechanism by which inflammation alters osteoblast differentiation and fate decision via the IKK2/mTOR/glycolysis axis.
Abstract Bone remodeling requires precise coordination between osteoblast-mediated bone formation and osteoclast-driven resorption. However, directly targetable and therapeutically actionable mediators that can coordinately modulate both processes during regeneration remain relatively limited. Here, we identify immunoglobulin superfamily member 10 (IGSF10) as a dual-function modulator of bone remodeling. Igsf10- deficient mice exhibit reduced bone mass, elevated osteoclast activity, and impaired osteogenesis. Recombinant IGSF10 protein restores osteogenic capacity and suppresses osteoclastogenesis in knockout cells, while exerting pro-osteogenic and anti-resorptive effects in wild-type mesenchymal and monocyte-derived cultures. Mechanistically, IGSF10 activates a noncanonical EGFR–STAT1 signaling pathway, distinct from BMP2–Smad signaling. Co-immunoprecipitation and molecular docking confirm IGSF10–EGFR interaction, and blockade of EGFR abrogates IGSF10-induced osteogenesis and its inhibitory effects on osteoclastogenesis. In vivo, IGSF10 promotes bone regeneration in both calvarial and periodontal defect models and exhibits synergy with subtherapeutic BMP2. These findings position IGSF10 as a previously unrecognized dual-acting regulator that coordinates bone formation and resorption in a context-dependent manner, with potential therapeutic value for craniofacial and skeletal regeneration.
During homeostasis, osteocyte apoptosis is typically associated with bone loss through enhanced osteoclast recruitment and bone resorption. However, whether apoptotic osteocytes also regulate bone formation remains elusive. Here we report that conditional deletion of Mdm2, an E3 ubiquitin ligase regulating cell survival, causes osteocyte apoptosis but paradoxically results in a marked increase in bone mass attributed to up-regulated osteogenic activity. Single-cell RNA sequencing reveals enhanced osteoblastic differentiation of bone marrow mesenchymal stem cells (BMSCs) in conditional knockout mice, with enrichment of cellular calcium related pathways. Mechanistically, apoptotic vesicles (apovs) from Mdm2-deleted osteocytes are engulfed by BMSCs. TRPM8, a calcium channel protein, is enriched in osteocyte-derived apovs and transported into BMSCs, thereby promoting osteoblastic differentiation. Additionally, pharmacological inhibition of TRPM8 attenuates the high bone mass phenotype in conditional knockout mice. Therefore, Mdm2 deletion in osteocytes leads to osteocyte apoptosis, which enhances bone formation through communicating with BMSCs via TRPM8-enriched apovs. Our findings underscore the pivotal role of osteocytes in bone homeostasis and unveil a previously unrecognized mechanism whereby osteocyte apoptosis stimulates osteogenesis through affecting the fate of BMSCs in a TRPM8-mediated paracrine mechanism.
Osteogenesis imperfecta (OI) is a rare bone fragility disorder. Previously, in a severe OI mouse model (Col1a1Jrt/+), a sex- and age-dependent metabolic phenotype was observed, correlating with elevated levels of the bone-derived hormone osteocalcin (OCN). This hormone is known to play a crucial role in managing energy metabolism, including glucose regulation and fat mass. In fact, upon high-fat diet (HFD) exposure, OI mice developed a metabolic syndrome linked to sex and OCN. To assess OCN’s role in OI, Col1a1Jrt/+ mice were crossed with OCN-deficient mice (Bglap). Under regular chow and HFD conditions, both OCN-dependent and OCN-independent metabolic alterations were identified. OCN-dependent processes were adipose tissue, liver, and insulin metabolism in a sex-, age-, and diet-dependent manner. OCN-independent traits included the pancreas in juvenile mice, HFD-induced pancreatic insulin levels and glucose intolerance, besides overall growth, fertility, and bone phenotype. Notably, increased juvenile energy expenditure was OCN-independent, while HFD-induced changes were OCN-driven. These findings demonstrate OCN’s role in shaping the metabolic phenotype while revealing distinct OCN-independent effects, emphasizing the complex genetic regulation of metabolism in OI.
Bone healing is orchestrated by multiple cell populations, including those that normally do not produce bone but acquire osteoprogenitor capabilities following injury. Muscle-resident fibroadipogenic progenitors (FAPs) and superficial periosteal cells are among such cells, however their physiologic significance in bone healing and the mechanisms regulating their differentiation are unclear. Here, using the new tamoxifen-inducible Clec3b.CreERT2 allele, we show that both of these populations can be traced and manipulated in a mouse model. Clec3b+ cells are completely absent in marrow and bone-lining surfaces and can differentiate to multiple cell types including osteoblasts during fracture repair and BMP2-induced heterotopic ossification. Orthotopic transplantation assays indicate that Clec3b+ FAPs rather than Clec3b+ periosteal cells are mobilized to become osteoblasts in these conditions. Further, Clec3b+ cells appear functionally distinct from periosteal skeletal progenitors as they exhibit remarkably low chondrogenesis during fracture healing; however, they can form cartilage during heterotopic ossification of muscle and ex vivo culture. Inhibition of WNT-signaling or depletion of Clec3b+ cells reduce mineralization during both processes. These data show that extra-skeletal cells that normally do not produce bone, FAPs in particular, are recruited to help repair bone fractures, and could represent a novel target for therapies aimed to enhance fracture healing.
The core pathological mechanism of steroid-induced osteonecrosis of the femoral head (SONFH) is an “immune freeze” microenvironment—where corticosteroids continuously drive macrophages toward a pro-inflammatory M1 phenotype, inhibiting the conversion to reparative M2 macrophages, thereby impairing bone regeneration. To address this bottleneck, this study was inspired by the hierarchical pore structure of coral. Using low-temperature deposition 3D printing technology, we constructed an immunoreprogramming biomimetic scaffold that integrates multi-walled carbon nanotubes (MWCNT) and nano-hydroxyapatite (nHA) (MWCNT bionic scaffold). The scaffold leverages the active immune regulatory function of MWCNT to activate the PI3K-AKT signaling pathway, driving macrophages to transition from the M1 to M2 phenotype, effectively breaking the “immunological freeze” state and reshaping the pro-regenerative bone immune microenvironment. Meanwhile, the nHA component provides a biomimetic mineralization matrix and sustained release of calcium and phosphate ions, synergizing with the nanofiber structure of MWCNT to promote the migration and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and angiogenesis. In vivo experiments confirm that scaffold implantation reverses the local “immune freeze” state, drives macrophage polarization toward the M2 phenotype, reduces inflammatory responses, and enhances the maturation and functional vascular network formation of new bone matrix in bone defect areas, ultimately achieving bone structural reconstruction. The coral-inspired immunoreprogramming strategy proposed in this study provides a new strategy for targeting the regulation of the pathological microenvironment in SONFH.
Cell-cell fusion, essential for diverse physiological events, requires high ATP levels. While mitochondrial activity increases in fusing cells, the mechanism driving mitochondrial ribosome (mitoribosome) biogenesis to support these energy demands remains unclear. Here, we identify angiogenin (ANG) as a mitochondrial tRNA (mt-tRNA) processing enzyme critical for mitoribosome biogenesis during myoblast and osteoclast fusion. Upon fusion initiation, ANG translocates to mitochondria, promoting mitoribosome biogenesis to support translation of respiratory complex proteins for ATP production. Using transcriptome-wide PARE and 5' RACE analyses, we show that ANG cleaves the tRNA 3'-end in mitochondrial pre-RNA transcripts bordering rRNAs and mRNAs, enabling their release for translation. Loss of ANG or disruption of its ribonucleolytic activity impairs osteoclast and myoblast fusion, disrupting bone and muscle homeostasis and skeletal muscle regeneration post-injury. Our findings establish ANG as an essential mitoribosome biogenesis regulator and highlight a novel mechanism of mitochondria energy regulation in high-energy-demand biological processes.
Abstract Osteogenesis imperfecta (OI), characterized by bone fragility and low bone mass, is predominantly caused by mutations in type I collagen. High bone mass OI (HBM OI) is a rare form caused by heterozygous missense mutations at the type I procollagen C-propeptide cleavage site. Knock-in HBM OI mice were generated to elucidate the effect of this mutation on cells and bone. HBM OI murine femora contain increased monomeric pro-α1(I)C-propeptide and pC-collagen; their bone collagen fibrils have a “barbed-wire” appearance. Decreased C-propeptide cleavage diminishes bone strength. HBM OI femora are extremely brittle, with thin cortices, decreased BV/TV, and fracture load. The cortical bone has increased mineral content, with thinner, more disorganized mineral particles. Increased expression of ossification genes in both murine and human HBM OI osteoblasts during in vitro differentiation and increased mineral deposition in culture indicate impaired C-propeptide processing affects cellular processes related to mineralization, rather than being a passive matrix process. Gene ontology analysis of RNA-seq data from differentiating HBM OI osteoblasts revealed top upregulated pathways for ossification, mineralization, and osteoblast differentiation (5–25×) while top-downregulated pathways involved cellular adhesion, migration, and angiogenesis (5–10×), all related to cell-matrix interactions. Moreover, the HBM matrix affects osteoblast function. WT osteoblasts plated on HBM OI decellularized matrix in vitro showed less punctate vinculin, increased peripheral actin staining, and the presence of lamellipodia, suggesting a decrease in cellular adhesion. Insights into the mechanism of HBM OI mineralization may lead to improved therapies for HBM OI and low bone mass conditions.
Stromal progenitor cells of bone marrow origin are non-hematopoietic cells that give rise to osteoblasts and adipocytes in the postnatal organism. Marrow stromal cells (also known as mesenchymal stem cells - MSCs) are currently being employed in a large number of clinical trials for regenerative purposes post in vitro expansion. However, the clinical outcome has been variable, which might in part be due to the heterogeneity of the cells and the lack of a defined cell product with a molecular signature that favors tissue regeneration. In this study, we determined the cellular heterogeneity of primary stromal cultures and examined how inter-donor variation in subpopulation composition contributes to the differentiation potential of primary cultures. We profiled 136 014 stromal progenitors from 26 donors and identified 5 subpopulations that were linked to distinct bone-related pathways and genetic traits of bone mineral density and morphology. Abundance of one cluster characterized by high expression of ITGA11 (integrin alpha-11) and genes related to matrix function, collagen organization, and elevated expression upon lineage commitment was positively correlated with osteoblastic differentiation capacity in vitro. In addition, ITGA11 protein expression in progenitor cells was a predictive marker for matrix mineralization in vitro and ectopic bone formation in vivo. Sorting stromal progenitors into ITGA11high and ITGA11low cells established cultures with high and low osteoblastic differentiation potential and revealed transcriptional differences reflective of the subpopulation-specific signature, which was not affected by siRNA-mediated knockdown of ITGA11 expression. Our findings corroborate the presence of an extensive donor-dependent cellular heterogeneity that persists in cultured stromal cells, and that ITGA11 can be employed as a marker for isolating cells with high bone-forming potential, a feature likely to benefit clinical trials of bone regeneration.
Osteoarthritis (OA) is a prevalent chronic degenerative joint disease that significantly impacts the quality of life for over 500 million individuals affected. OA is increasingly recognized as a whole-joint disorder characterized by complex biochemical and cellular changes across various joint tissues. However, traditional approaches such as nonsteroidal anti-inflammatory drugs (NSAIDs) and corticosteroids often fall short in halting disease progression or restoring joint function. Thereby, recent innovations in OA management are focused on underlying pathophysiology, driving the development of regenerative therapies, targeted anti-inflammatory agents and senolytic or senomorphic approaches. Accordingly, this paper reviews current progress in these areas by integrating evidence from clinical and pre-clinical studies to clarify therapeutic limitations, unresolved mechanistic and phenotypic gaps and the emerging strategies required to advance disease-modifying therapy in OA. It further outlines pathways toward precision, phenotype-aligned interventions, an integration that current literature has not yet consolidated.
