Peptide-Based Biomaterials for Bone and Cartilage Regeneration

The healing of osteochondral defects (OCDs) that result from injury, osteochondritis, or osteoarthritis and bear lesions in the cartilage and bone, pain, and loss of joint function in middle- and old-age individuals presents challenges to clinical practitioners because of non-regenerative cartilage and the limitations of current therapies. Bioactive peptide-based osteochondral (OC) tissue regeneration is becoming more popular because it does not have the immunogenicity, misfolding, or denaturation problems associated with original proteins. Periodically, reviews are published on the regeneration of bone and cartilage separately; however, none of them addressed the simultaneous healing of these tissues in the complicated heterogeneous environment of the osteochondral (OC) interface. As regulators of cell adhesion, proliferation, differentiation, angiogenesis, immunomodulation, and antibacterial activity, potential therapeutic strategies for OCDs utilizing bone and cartilage-specific peptides should be examined and investigated. The main goal of this review was to study how they contribute to the healing of OCDs, either alone or in conjunction with other peptides and biomaterials.


Introduction
An articulating joint's osteochondral (OC) unit comprises the vascularized and mineralized subchondral bone and the acellular and avascular hyaline cartilage connected by a seamless interface [1].The structural heterogenicity of the OC tissue arises from its diverse organic (cells, aggrecan, collagen) and inorganic (hydroxyapatite) components, along with their spatial orientation, forming gradients from the superficial cartilage to the subchondral bone via middle, deeper, and calcified layers (Figure 1).Cartilage exerts cushioning effects on joint bones and prevents damage from the physiological load; however, an injured cartilage cannot spontaneously regenerate.OCDs are created via a variety of biological (aging, osteochondritis, osteoarthritis) or mechanical (accidental trauma, sports injury, prolonged wear) factors and are characterized by cartilage and bone lesions, severe joint pain, and loss of joint function.They are more common in the middle-aged and older population.The World Health Organization estimates that 595 million people worldwide, or 7.6 percent of the world's population, suffered from osteoarthritis alone in 2020.This number has increased by 132.2 percent since 1990 [2].
Unfortunately, none of the existing procedures can fully repair an OCD or form hyaline cartilage instead of fibrocartilage [3][4][5][6][7].OC tissue engineering (TE) seems promising for regenerating functional OC tissue [8].Growth factors can promote MSCs, osteoblast or chondrocyte recruitment, proliferation and differentiation, vascularization, and maintenance of cartilage homeostasis.However, regulating their target selectivity, dose selection, release kinetics, and spatial distribution is quite challenging.Bioactive peptides are shortchain amino acid sequences that mimic the signaling or binding domains of larger proteins This review's primary objective was to find promising peptides or peptideconjugated biomaterials that will help to design a novel multi-peptide-based therapeutic strategy for rapid OCD healing, which has never been done before.After providing a brief overview of the OC unit and the requirements for OC regeneration, this study examined a wide range of bioactive peptides and peptide-conjugated biomaterials that have been reported to have osteo-, angio-, or chondrogenic potential for in vitro and in vivo bone and cartilage regeneration (Table 1).It also covers peptides that help OC regeneration by promoting cell migration and adhesion, assembling into a three-dimensional extracellular environment, or preventing an infection and immunological response.Finally, the review concludes with a summary and outlook.This review's primary objective was to find promising peptides or peptide-conjugated biomaterials that will help to design a novel multi-peptide-based therapeutic strategy for rapid OCD healing, which has never been done before.After providing a brief overview of the OC unit and the requirements for OC regeneration, this study examined a wide range of bioactive peptides and peptide-conjugated biomaterials that have been reported to have osteo-, angio-, or chondrogenic potential for in vitro and in vivo bone and cartilage regeneration (Table 1).It also covers peptides that help OC regeneration by promoting cell migration and adhesion, assembling into a three-dimensional extracellular environment, or preventing an infection and immunological response.Finally, the review concludes with a summary and outlook.Ti implant Osteogenic differentiation of BMSCs, mineralization in rat [14] GTPGQGIAGQRGVV (P15 peptide) HAP scaffold ↑ Osteogenic effect in rat [15] ((PKG) 4 -(POG) 4 -(DOG) 4 ) (KOD peptide) Peptide scaffold ↑ GAG and Col deposition in rabbit [16] DGEA PA scaffold Osteogenic differentiation of hMSC [17] NGLPGPIGP (BCSP™-1) Injection In vitro mineralization of bone marrow-derived osteoblasts and in rats [18] GPAGPHGPVG, APDPFRMY, TPERYY Culture medium ↑ Osteogenic activity in MC3T3-E1 cells [

Requirements for Osteochondral Tissue Regeneration
The prerequisites of OC regeneration are native tissue mimetic scaffolds, reproducing the heterogeneous structure of the OC tissue, and a well-controlled inflammatory state to counter MMPs-mediated irreversible destruction of the cartilage matrix.The plateletrich plasma (PRP) and exosome-based therapies, stem cell therapy [98], recombinant IL-1 receptor antagonists, and monoclonal antibodies against tumor necrosis factor-α (TNF-α)based therapy and genetic engineering have produced variable results.
On the other hand, tissue engineering, injectable hydrogels, and monolayer scaffolds fail to replicate the intricate spatial distribution of biochemical cues, particularly at the bone and cartilage interface [99,100].Advanced techniques, such as 3D printing or 3D bioprinting, enable the fabrication of customized scaffolds with structural anisotropy and heterogeneous cell distribution; however, they involve a high cost and intricate setup [101].Despite their osteogenic and chondrogenic activity, BMPs have less control over chondro-cyte hypertrophy and mineralization [98,102].The localized delivery of bioactive peptides to induce target protein expression; attachment and motility of particular cells; site-specific differentiation; and resist bacterial colonization at the defect site, inflammation, or immunogenicity could be an ideal choice for biomimetic bone and cartilage regeneration.
On the other hand, tissue engineering, injectable hydrogels, and monolayer scaffolds fail to replicate the intricate spatial distribution of biochemical cues, particularly at the bone and cartilage interface [99,100].Advanced techniques, such as 3D printing or 3D bioprinting, enable the fabrication of customized scaffolds with structural anisotropy and heterogeneous cell distribution; however, they involve a high cost and intricate setup [101].Despite their osteogenic and chondrogenic activity, BMPs have less control over chondrocyte hypertrophy and mineralization [98,102].The localized delivery of bioactive peptides to induce target protein expression; attachment and motility of particular cells; site-specific differentiation; and resist bacterial colonization at the defect site, inflammation, or immunogenicity could be an ideal choice for biomimetic bone and cartilage regeneration.
Collagen-mimetic GFOGER peptide, which is derived from the collagen α1 chain, selectively promotes α2β1 integrin binding required for osteoblastic differentiation [14].Besides improving cell attachment, GFOGER successfully induces in vitro osteogenic differentiation and in vivo bone healing [14,67,[108][109][110].GFOGER coating on synthetic PCL scaffolds remarkably enhanced bone formation in critically sized segmental defects in rats by stimulating osteoblast adhesion and differentiation (Figure 2b) [108].P15, which is a 15-mer peptide derived from the collagen type I α1 chain, has a strong affinity for the cell surface α2β1 integrin receptors.By releasing growth factors and cytokines, the peptide dramatically enhanced the osteogenic differentiation of MSCs [15].The commercially available P15 formulations significantly enhanced the regeneration of alveolar bone and tibial defects in osteoporotic dogs [111,112].The collagen-mimetic KOD peptide, which is made of three units, namely, ((PKG) 4 -(POG) 4 -(DOG) 4 ), forms a hydrogel through self-assembly inducing platelet activation and blood clotting associated with hematoma formation [113].POG-based poly-amphiphilic hydrogels allowed for faster recovery (within two weeks) of intervertebral disc defects in rabbits due to a significant increase in ECM deposition [16].DGEA derived from collagen type I adhesive motif serves as a crucial ligand for osteoblast differentiation [114].DGEA-containing PA hydrogels seeded with hMSCs substantially upregulated osteogenic markers (OCN, RUNX2, and ALP) [17].
Using a bone-and cartilage-stimulating peptide (BCSP™-1 or NGLPGPIGP) present in human collagen type-I, the proliferation of rat bone-marrow-derived osteoblasts and human or bovine chondrocytes was drastically improved with enhanced the bone mineral density (BMD) and bone mineral content (BMC) in male Wistar rats [18].Three highly osteogenic peptides (GPAGPHGPVG, APDPFRMY, and TPERYY) derived from tilapia scale collagen hydrolysate notably increased the MC3T3-E1 cell proliferation and mineralization activity (ALP synthesis, osteogenic-related gene expression) at concentrations of 50 µg/mL [19].

BMP-Mimetic/Derived Peptides
As members of the TGF-β superfamily, BMPs are primarily produced by endothelial cells (ECs), osteoblasts, and hypertrophic chondrocytes and can recruit MSCs to the site of injury and differentiate into osteoblasts while inducing ectopic bone formation.

Circulating Peptides
Osteogenic growth peptide (OGP), which is a 14-mer peptide occurring in mammalian blood, increases bone formation through anabolic effects on bone cells [131,132] and differentiation of osteoprogenitor cells, leading to upregulated osteogenic markers, including mineralization [36][37][38].Thrombin peptide 508 (TP508) or Chrysalin is a 23-amino acid peptide and receptor binding domain of thrombin, which enhanced the proliferation, differentiation, and chemotaxis of human osteoblasts [39,40] and VEGF-stimulated angiogenesis [133].TP508 injected into the fracture gap promoted fracture healing and increased blood vessel formation [61,134,135].
Collagen-binding motif (CBM) is a cleavage product of osteopontin (OPN) that can specifically bind to collagen [138] and promote migration, osteogenic differentiation [139], and bone formation in a rabbit calvarial defect model [42].The SVVYGLR peptide adjacent to the RGD sequence in OPN significantly enhanced the adhesion and proliferation of MSCs, neovascularization, upregulation of osteogenesis, and angiogenesis when delivered through a collagen sponge [43][44][45].FHRRIKA, which is a cell-binding and heparin-binding domain of bone sialoprotein (BSP) exerts a favorable effect on osteoblast adhesion, spreading, and mineralization [46].Higher cell proliferation and viability were observed on rat calvarial osteoblasts that seeded scaffolds containing the RGD and FHRRIKA sequences [140].

Biomineralizing Peptides
Non-collagenous proteins (NCPs), such as dentin sialophosphoprotein, dentin matrix protein 1 (DMP1), and dentin phosphoprotein (DPP), play a significant role in biomineralization.The negatively charged domains (carboxylic acid and phosphate groups) in NCPs serve as preferential sites for the nucleation of hydroxyapatites (HAPs) while stabilizing them into the self-assembled collagen fibrils that act as a template for crystal growth.Peptides derived from such proteins significantly enhance bone formation.
The Asp-Ser-Ser (DSS) repeating motifs present in DPP have a remarkably strong binding affinity toward calcium ions and HAP [141].8DSS, which is a DPP peptide with eight repetitive units of DSS, was the most promising for promoting the mineralization and remineralization of acid-etched enamel [47,48].Like DSS, 3NSS with three repetitive units of asparagine-serine-serine (aspartic acid in DSS is substituted with asparagine) could remineralize the acid-etched enamel [49].On the other hand, the DSESSEEDR sequence in dentin matrix protein 1 (DMP1) could bind to demineralized dentin and promote remineralization [50].The other phosphoprotein-derived peptides, such as SN15, SNA15, DpSpSEEKC, DDDEEK, and DDDEEKC, exhibited high affinity toward HAP [142].
Amelogenin, which is found at the dentin-enamel interface, interacts with collagen to control the formation of HAP crystals and their structural alignment [143].In addition to remineralizing enamel caries, amelogenin-inspired peptides, such as shADP5, QP5, P26, and P32, helped to restore demineralized dentin [51,52].Since leucine-rich amelogenin peptide (LRAP) is more hydrophilic than amelogenin, the demineralized enamel treated with CS-LRAP hydrogel exhibited quicker nucleation and development of HAP crystals than amelogenin-containing chitosan hydrogel (CS-AMEL) [53].A non-amelogenin protein called tuftelin is present in tooth enamel and has a role in the mineralization of dental enamel.Tuftelin-derived peptide (TDP) encouraged the remineralization of early carious lesions by attracting calcium and phosphate ions [54].Self-assembling amphiphilic oligopeptide derived from cementum protein 1 (CEMP1), which is a regulator of cementum-matrix mineralization, induced intrafibrillar mineralization of collagen fibrils in the presence of calcium ions [55].P11-4, which is another self-assembling peptide, acted as a scaffold to enhance HAP nucleation de novo [56].

Angiogenic Peptides
Vascularization is a crucial process during natural bone formation.Many peptides are derived from angiogenic growth factors (e.g., VEGF, fibroblast growth factor-2 (FGF-2), and PDGF), ECM (e.g., OPN, ON), and other proteins that have crucial roles in blood vessel formation [144].
VEGF-mimicking QK or KLT peptide (KLTWQELQLKYKGIGGG), which is derived from the VEGF receptors binding domain 17-35, not only induces EC migration and proliferation but also triggers other complex processes, like chemotaxis and capillary sprouting and organization similar to VEGF [57].PDGF-BB-derived PBA2-1c peptide interacts with αand β-PDGF receptors.Though its in vivo proangiogenic activity is still unclear, it functions similarly to PDGF in establishing mature blood vessels that are created by VEGF [58].Exendin-4, which is a glucagon-like peptide 1 (Glp-1) analog, stimulates human umbilical vein endothelial cells' (HUVECs') motility, sprouting, and tube formation in vitro, in addition to in vivo sprout outgrowth [59].While OPN is widely distributed in the bone matrix to help with bone metabolism, OPN-derived peptide (OPD) does not induce EC proliferation in vitro.However, like VEGF, it facilitated EC migration and tube formation using 3D collagen gels [145], suppressed osteoclastogenesis [43], and promoted the adhesion and proliferation of MSCs, as well as neovascularization in a rat tibial defect model [146].SPARC113 and SPARC118, which are two OPN-derived peptides that exhibit potent angiogenic activity [147], stimulated in vivo angiogenesis when delivered through MMPs degradable hydrogel [60].TP508 enhanced neoangiogenesis in femoral defects produced in rats [61] and mice [134] following one hour of local administration.The synthetic 12-mer peptides, known as RoY peptides, which were created via the phage-display technology, may also promote in vitro EC proliferation, tube formation, and sprouting, as well as induce in vivo angiogenesis via a distinct mechanism from VEGF [62].

Chondroinductive/Chondrogenic Peptides
Numerous peptides were identified to imitate the functions of ECM components, cellcell junction molecules, and chondroinductive/chondrogenic ligands triggering specific cell-signaling pathways.Motif-derived fibronectins, like RGD, decorin, collagen, and MMPs, display chondrogenic properties.These peptides are often used to functionalize scaffolds that encourage chondrocyte adhesion, migration, and proliferation, in addition to MSC differentiation into the chondrogenic lineage.

Adhesion, Binding, or Affinity Peptides
RGD peptide: The RGD (Arg-Gly-Asp) motif is an essential cell adhesion peptide found in collagen, FN, vitronectin (VN), etc., that promotes cell adhesion and/or differentiation through binding with transmembrane integrin receptors (Figure 3a).RGD sequences, such as GRGDS, RGDS, YRGDS, and c(RGDfk), are widely used in the field of cartilage and bone repair.Bone regeneration using cyclic RGD in a sheep spinal fusion model was comparable with that of rhBMP-2 due to its increased receptor affinity [66].RGD, particularly the longer chain RGD (GPenGRGDSPCA), was not always effective in increasing cell adhesion and spreading.This could be because pre-absorbed proteins with stronger binding sites (like FN and VN) compete with weekly adsorbed RGD for cell binding, necessitating covalent attachment [69,154].RGD is usually capable of increasing the cellular adhesion of MSCs but is unable to induce osteogenic differentiation without osteogenic supplements or BMP-2 (Figure 3b) [109,116,117].
RGD, particularly the longer chain RGD (GPenGRGDSPCA), was not always effective in increasing cell adhesion and spreading.This could be because pre-absorbed proteins with stronger binding sites (like FN and VN) compete with weekly adsorbed RGD for cell binding, necessitating covalent attachment [69,154].RGD is usually capable of increasing the cellular adhesion of MSCs but is unable to induce osteogenic differentiation without osteogenic supplements or BMP-2 (Figure 3b) [109,116,117].PHSRN peptide: PHSRN is an FN-derived peptide that synergistically enhances cellular activity with RGD and other adhesion peptides through α5β1 integrin binding, though it is ineffective alone [67,68].Due to competitive binding with α5β1 integrin sites, RGD and PHSRN did not improve the spreading of rat bone MSC on titanium surfaces [156].However, a dimeric platform consisting of RGD and PHSRN, with a spacing between them resembling natural fibronectin, significantly improved the spreading and proliferation of SAOS-2 osteoblast-like cells when compared with RGD alone or their PHSRN peptide: PHSRN is an FN-derived peptide that synergistically enhances cellular activity with RGD and other adhesion peptides through α5β1 integrin binding, though it is ineffective alone [67,68].Due to competitive binding with α5β1 integrin sites, RGD and PHSRN did not improve the spreading of rat bone MSC on titanium surfaces [156].However, a dimeric platform consisting of RGD and PHSRN, with a spacing between them resembling natural fibronectin, significantly improved the spreading and proliferation of SAOS-2 osteoblast-like cells when compared with RGD alone or their combination [68].Further improvements in attachment, spreading, and cell differentiation were made possible by a longer-chain RGD that allowed for wider spacings between the peptides like natural fibronectin; nevertheless, such modifications did not result in any osteogenic activity [157].
Affinity peptides can bind with specific cells, scaffolds, and cytokines associated with cartilage regeneration.Using an anti-CD44 antibody, biotin-avidin binding system [163], chondrocyte affinity peptides [164], E7 peptide [165], etc., cell adhesion was greatly improved.A TGF-β affinity peptide that can recruit TGF-β to the impaired region was ligated to the PA nanofibers and promoted in vitro chondrogenic differentiation of hMSCs and cartilage repair in rabbits [166,167].ECM affinity peptides mimicking the native environment of chondrocytes were integrated with scaffolds [168,169].Hydrogels modified with HA and chondroitin sulfate-binding peptides significantly promoted BMSC differentiation into a chondrogenic lineage (Figure 3c) [86].
While RGD peptides immobilized at a low density effectively promoted cell adhesion and chondrogenesis [170][171][172], a higher density of RGD induced the hypertrophic transformation of chondrocytes due to the activation of the integrin-mediated pathway [173][174][175].However, compared with simple RGD adsorption, the chemical grafting of RGD peptides onto the biomaterial surface offered improved coating stability and osteoconductivity (Figure 3d) [155].The incorporation of the N-cadherin-mimetic HAVDIGGGC peptide into HA hydrogels increased chondrogenic differentiation [176,177].Similarly, laminin-related CDPGYIGSR peptide significantly promoted the adhesion of bovine knee chondrocytes onto the polyethylene oxide PEO/chitosan scaffolds [78].

Peptides Promoting Cell Migration
Migration of fibroblasts, MSCs, osteoblasts, chondrocytes, and EC toward the defect site is required for OCD healing.ECM proteins and GFs (EGF, PDGF) act as mitogens or chemotactic factors facilitating cell migration [181].Peptides derived from them often exhibit superior cell migration than the parent protein.Stromal cell-derived factor-1α (SDF-1α) is a known recruiter of endothelial progenitor cells during osteochondral repair [182].SDF-1-derived elastin-like peptide (SDF1-ELP) showed comparable EC migration and vascularization to SDF and even more effective healing of full-thickness diabetic wounds in mice [79].Histatin-1, which is a 38-mer antimicrobial peptide found in saliva, exerts a proangiogenic effect that stimulates the adhesion and migration of EC [80].The endogenous histatins, annexin A1-derived peptide Ac2-26 [81], or frog-skin-derived esculentin 1-21 peptide [82] showed better wound-healing activity driven by cell migration.The administration of LL-37 significantly increased vascularization because of enhanced EC migration [183].Like FGF-2 (bFGF), the FGF-2-mimicking peptide YRSRKYSSWYVALKR derived from the 106-120 peptide domain in FGF-2, which is a partial agonist of FGF receptors, could successfully promote in vitro HUVEC cells proliferation and migration [184].

Self-Assembly (SA) Peptides
SA peptides are composed of either alternating hydrophilic and hydrophobic amino acids or peptide amphiphiles (PA), which can self-assemble into β-sheet structures and interwoven nanofibrous hydrogel matrices under physiological conditions [185][186][187].SA peptides isolated from various growth factors and bone-related proteins (RANKL-binding peptide, AC-100, B2A) were studied for osteoblast differentiation and bone regeneration [21,188,189].A RADA16-I peptide immobilized onto the BMP-2 loaded hydrogel promoted osteogenic differentiation of MSCs, leading to a higher expression of osteogenic-related genes (ALP, OCN, Runx 2 ) and in vivo bone regeneration [85].PuraMatrix TM , which is a commercially available RADA16-1-peptide-containing hydrogel, strongly supported cartilage formation [190,191].Repeating units of KLD and RAD in a peptide hydrogel enhanced cartilage formation compared with an agarose gel [192].

Degradable Peptides
Degradable peptides accelerate the degradation of scaffolds to facilitate cell penetration and migration, ECM synthesis, and deposition, and consequently, tissue ingrowth.MMPs are the best examples of ECM-degrading proteins.The inclusion of an MMP-derived peptide (KCGPQGIWGQCK) remarkably promoted GAGs and collagen deposition onto crosslinked poly(ethylene glycol) norbornene hydrogels [86,87].
AMPs work either by disrupting the bacterial cell wall or cytoplasmic membrane (Figure 4a) or by interfering with cell wall synthesis.The bactericidal properties of keratinderived anti-inflammatory KAMP-19 peptide originate from the destruction of the bacterial cell membrane via pore formation [90].α-defensin, which is known as human Neutrophil Peptide 1 (HNP-1), through its high affinity for lipid II, destabilizes the cell wall integrity [91].Likewise, RWRWRW-NH 2 delocalizes peripheral membrane proteins [92], LL-37 affects the Sfp1 gene associated with cell wall synthesis, and buforin II and TO17 interfere with nucleic acid functions [89,194].
AMPs work either by disrupting the bacterial cell wall or cytoplasmic membrane (Figure 4a) or by interfering with cell wall synthesis.The bactericidal properties of keratinderived anti-inflammatory KAMP-19 peptide originate from the destruction of the bacterial cell membrane via pore formation [90].α-defensin, which is known as human Neutrophil Peptide 1 (HNP-1), through its high affinity for lipid II, destabilizes the cell wall integrity [91].Likewise, RWRWRW-NH2 delocalizes peripheral membrane proteins [92], LL-37 affects the Sfp1 gene associated with cell wall synthesis, and buforin II and TO17 interfere with nucleic acid functions [89,194].Biofilms associated with chronic bone infection are difficult to treat because of bacterial resistance [197].LL-37 interacts with the cell wall component mannan to lower Candida adhesion (Figure 4b) [195].AMP 1037, which is a 9-mer peptide, decreases the swimming and motility of bacteria [93].Piscidin and sculentin (1-21) degrade a pre-formed biofilm matrix by activating nuclease activity and disturbing the membrane function, respectively [94,95].
Anti-inflammatory cytokines (AMPs) can suppress pro-inflammatory cytokine signaling pathways or stimulate the synthesis of anti-inflammatory cytokines associated with an innate and adaptive immune response (Figure 4c).Immunomodulatory metalloproteinases (AMPs), such as cathelicidin-WA, stimulate macrophage polarization from the M1 phenotype (caused by E. coli) to the M2 phenotype (facilitating bone repair) (Figure 4d) [196].Macrophage polarization plays a significant role in mediating chondrogenesis.Failure in the M1 to M2 transformation may lead to the progression of cartilage injury and interrupted tissue remodeling and repair [198,199].

Peptide-Conjugated Biomaterials
Various peptides with osteo-or chondrogenic properties were delivered or conjugated with synthetic and natural biomaterials for better OCD healing.

Osteo-Inductive Scaffold
Delivery of the BMP2-derived KIPKASSVPTELSAISTLYL peptide through TCP scaffolds promoted bone healing in rabbit radial bone defects [201].BMP-2 mimetic peptides attached to a substrate enhanced the BMSC attachment and differentiation without affecting the mineralization [117].However, when these peptides were combined with RGD, the rate of mineralization increased because RGD induced higher interaction with cells in the absence of osteogenic supplementation [116].The incorporation of BMP-2-mimetic peptide conjugated to a heptaglutamate moiety (E7-BMP-2) into the mineralized PLGA-collagengelatin nanofiber promoted bone formation in critical-sized alveolar bone defects in rats after 4 weeks of implantation [202].An electrospun porous cellulose acetate nanofibrous mat modified with adhesive peptides KRSR, RGD, and growth factor BMP-2 enhanced the adhesion and proliferation of pre-osteoblastic cells [203].PCL nanofibrous scaffolds containing different concentrations of the bioactive with the KIPKASSVPTELSAISTLYL peptide derived from the BMP-2 scaffolds developed by Lukasova et al. and seeded with porcine MSCs showed an increased expression of OCN, and collagen I significantly increased osteogenic differentiation [204].KRSR, BMP mimetic peptides, and FHRIKKA were successfully integrated into the outer, middle, and inner layers of PLGA electrospun membranes through layer-by-layer assembly, and such modification demonstrated synergistic effects on bone healing by enhancing cell attachment, differentiation, and mineralization [205].FHRRIKA and KRSR peptides that were covalently linked with the surface of polymeric nanoparticles significantly improved MSC attachment and ALP activity due to osteogenic differentiation, which was not very profound in the case of KRSR [206,207].The immobilization of KRSR on a silane-functionalized borosilicate glass [158], micropatterning with KRSR on borosilicate glass [208], or another substrate [69,73,74,209] selectively enhanced osteoblast adhesion through αvβ5 integrin receptors binding.
On the other hand, GFOGER-functionalized PEG-BMP-2 hydrogels increased in vitro cell spreading and differentiation of BMSCs into an osteogenic lineage, as well as improved bone healing in a mice radial defect model, even in the absence of BMP-2 [109].Implants coated with GFOGER were found to improve peri-implant bone regeneration and osseointegration [210].However, hydroxyapatite disks adsorbed with GFOGER failed to attach cells with a decrease in cell spreading [154], possibly due to a lack of nature-mimicking conformation of GFOGER [108].P15-coated bovine bone grafts allowed for the faster recovery of periodontal defects in humans [211].P-15-containing bone graft substitutes could facilitate the process of early bone formation [212,213], bone healing, and regeneration [214].P24 delivery to the defect site was achieved by encapsulating in chitosan microspheres [118], covalent binding with polymeric backbone [22], or simple adsorption on hydroxyapatite [22], which greatly enhanced the bioactive properties of the scaffolds.
DGEA immobilized on HA was found to enhance the adhesion and osteoblastic differentiation of MSCs and new bone formation [215].BCSP™-1, when delivered using HA and tricalcium phosphate (TCP) grafts, stimulated ALP activity in murine calvarial osteoprogenitor cells [216].OGP-rich PLGA scaffold accelerated bone healing in 1.5 cm rabbit segmental defects [217].A PEG hydrogel containing PTH1-34 promoted in situ bone augmentation in rabbits [218], whereas TP508-loaded PPF composite and microsphere scaffolds showed enhanced bone formation in rabbit segmental bone defects [219].RGD-coated implants were found to have an increased peri-implant bone formation and enhanced direct bone apposition, even in areas of poor surrounding bone [155,[220][221][222]].An increased osteoblast density was observed when an anodized nanotubular titanium surface was coated with RGD or RGDS (Arg-Gly-Asp-Ser) [223,224].

Multifunctional Scaffolds
The subcutaneous injection of nanofibrous hollow microspheres conjugated with TGF-β1 mimetic CM10 combined with rabbit BMSCs successfully induced ectopic cartilage formation in nude mice via the chondrogenic differentiation of MSCs; in contrast, BMP-2 mimetic P24-peptide-loaded microspheres demonstrated osteogenic activity leading to bone formation [64].The implantation of 3D printed porous bi-layered scaffolds infiltrated with TGF-β1 binding HSNGLPLGG(MA)-peptide-rich GelMA hydrogels in the top layer and hydroxyapatite in the bottom layer successfully repaired the osteochondral defects in SD rats through simultaneous cartilage and bone regeneration, respectively [229].Hyaluronic-acid-based hydrogel particles (HGPs) functionalized with a cysteine-tagged CK2.1 peptide showed promising results for cartilage repair in a mouse model without inducing chondrocyte hypertrophy, unlike BMP-2 [28].Meanwhile, using the same model, CK2.2 and CK2.3 peptides induced osteoblast differentiation and mineralization, similar to BMP-2 [65].RGD-functionalized poly(ethylene glycol)-diacrylate (PEGDA) hydrogel encapsulating rat osteoblasts facilitated the adhesion and spreading of rat osteoblasts, leading to matrix mineralization [230].RGD immobilized on hydroxyapatite improved the healing of femoral condylar defects in rabbits [231].However, a longer chain RGD was unable to do the same without a TGF-β supplementation [232].RGD peptide attached to glass coverslips through silanization did not show osteoblast adhesion since attachment happened through integrin binding [158].
Wang et al. developed a cryogenic 3D printed bilayer β-tricalcium phosphate/PLGA osteochondral scaffold that was enriched with osteogenic peptide sequence, KIPKA SSVPT ELSAI STLYL SGGC, and TGF-β1-loaded collagen I hydrogel for cartilage repair.The osteochondral scaffold showed improved chondrogenic differentiation of rBMSCs via the upregulated expression of chondrogenic markers and glycosaminoglycan (GAG) production, while it also enhanced the osteogenic differentiation of rBMSCs at the subchondral layer via the upregulated expression of RUNX2, osteocalcin and alkaline phosphatase (ALP), and calcium deposition [233].
Due to the bactericidal or anti-biofilm, immunomodulatory, and regenerative capacity, AMPs with adequate cytocompatibility have been explored for bone and cartilage tissue regeneration.A titanium surface immobilized with Mel4 peptides demonstrated significant antimicrobial efficacy after implantation into the rabbit femoral defects [234].While a GL13K peptide (GKIIKLKASLKLL-NH 2 )-coated titanium surface could effectively inhibit peri-implantitis through immunomodulatory function [235,236], a titanium plate modified with antimicrobial LF1-11-coupled RGD peptide demonstrated good antibacterial properties with improved osteoblast adhesion, proliferation, and mineralization [237].The inclusion of HHC-36 (KRWWKWWRR), which is a broad-spectrum cationic AMP, and osteoconductive Laponite nanosilicates into GelMA hydrogel, which was used for coating of a titanium implant surface, significantly promoted the expression of osteogenic-related genes and mineralization of hMSCs, as well as effectively inhibited Gram-positive bacteria (S. aureus and S. epidermidis) and Gram-negative bacteria (P.aeruginosa and E. coli) [238].On the other hand, HA bone scaffolds coated with PSI10 (RRWPWWPWRR) and BMP2-MP [239] or a nano-hydroxyapatite coated titanium surface containing antimicrobial human β-defensin 3 (HBD-3) peptide and BMP-2 [240] significantly promoted the antibacterial and osteogenic activities of the scaffolds.
Multifunctional PEEK coated with lithium-ion and mussel-inspired antimicrobial peptide enhanced the osteogenesis-associated genes/proteins expression and osseointegration at the bone-implant interface, as well as the inhibition of E. coli and S. aureus [241].A coating with mouse beta-defensin-14 (MBD-14) also improved the antimicrobial and osseointegration properties of PEEK [242].A KR-12 analog immobilized on the surface of titanium or PEEK bone implants via a polydopamine coating showed remarkable antibacterial activity, osteogenic differentiation of MSCs, and peri-implant bone formation [243,244].
The adjuvant peptides (RGD, PHSRN, SDF1-ELP, NLS-TAT, RADA16-I, KCGPQGI-WGQCK) may support by facilitating cell adhesion, migration, intracellular cargo delivery, or the creation of a 3D microenvironment through self-assembly.Peptides such as LL-37, GL13K, KAMP-19, AMP 1037, and β defensin-3 can treat post-implantation issues like infection, inflammation, or an immunogenic reaction.In this context, pattern-based searching using artificial intelligence (AI) and machine learning (ML) tools can identify more unique sequences or bioactive motifs from full-length proteins.Considering the structural and functional heterogeneities of OC units, a strategy involving multiple bioactive peptides or combining them with biomaterials for simultaneous bone and cartilage regeneration could be fruitful.For instance, the blending of collagen-mimetic GFOGER or P15 peptides, or BMP-2 mimetic P24 or BFP-2 peptides, along with VEGF-mimetic QK, TP508, or RoY peptides, can synergistically promote osteo-inductive effects.
Scaffolds or hydrogels and other bone graft substitutes coupled with GFOGER, FHRIKKA, KRSR, P-15, DGEA, or adhesive peptides (RGD or PHSRN) may significantly improve osteogenic activity and in vivo bone formation.The delivery of HSNGLPL, KLD12, and KLD12-CMP7 peptides through pluronic F-127 hydrogel, chitosan, or poly(L-lactide-co-caprolactone) scaffolds may promote cartilage regeneration.Short SA peptides coassembled with macromolecules may enhance the mechanical and biological properties of hydrogel nanocomposites.Cationic AMPs, such as GL13K or HHC-36, were often valuable for enhanced osteogenic activity apart from their inherent antimicrobial or immunomodulatory properties.
Multifunctional GelMA/HA, PEGDA, β-TCP/PLGA scaffolds or hydrogels, PEEK, and titanium implants combined with osteo-and chondrogenic peptides may facilitate the simultaneous healing of OCDs.However, more study on electrospinning, inkjet printing, and bioprinting is required to accomplish the spatial distribution of multifunctional peptides within the constructs that resemble the intricate geometry and multi-scale hierarchy of native tissue.

Figure 1 .
Figure 1.Diagram of the osteochondral unit showing the structural hierarchy, zonal arrangements, and extracellular matrix composition of the articular cartilage.The cartilage matrix is mainly composed of collagen type II (Col II), hyaluronic acid (HA), aggrecan, and a large amount of water (H2O).Alignments of chondrocytes are flattened, spherical, and columnar in superficial, middle, and deep zones, respectively.On the other hand, chondrocytes are hypertrophic in the calcified matrix, primarily made of nanohydroxyapatite and collagen type I, and found inside lacunae.Proproline, Hyp-hydroxyproline, Gly-glycine.Figure generated via biorender.com.

Figure 1 .
Figure 1.Diagram of the osteochondral unit showing the structural hierarchy, zonal arrangements, and extracellular matrix composition of the articular cartilage.The cartilage matrix is mainly composed of collagen type II (Col II), hyaluronic acid (HA), aggrecan, and a large amount of water (H 2 O).Alignments of chondrocytes are flattened, spherical, and columnar in superficial, middle, and deep zones, respectively.On the other hand, chondrocytes are hypertrophic in the calcified matrix, primarily made of nanohydroxyapatite and collagen type I, and found inside lacunae.Pro-proline, Hyp-hydroxyproline, Gly-glycine.Figure generated via biorender.com.

Figure 2 .
Figure 2. Growth-factor-mimetic peptides for bone and cartilage regeneration in osteochondral defects.(a) Growth-factor-mimetic peptide epitopes inducing direct signaling, e.g., mimicking transforming growth factor-β (TGF-β) pathway for TGF-β receptors (TGFβR) binding (figure generated via biorender.com).(b) (i) Critical bone defects treated with GFOGER-coated scaffolds show significantly higher bone formation compared with uncoated PCL scaffolds and empty defect (control), as revealed using micro-CT imaging after 12 weeks of implantation; the plot also depicts a similar pattern at 4 weeks.(* Different from empty defect and PCL (p < 0.05)) (ii) Histology confirms that areas of high attenuation in GFOGER-coated sample, as revealed using 2D micro-CT image (inset) are bone tissue (red/pink) (soft tissue appearing blue/green in Sanderson's rapid bone stain) [108].Copyright© 2009 Elsevier Ltd.(c) Bone formation in mice treated with BFP-2 for 4 or 8 weeks in comparison with BMP-7 was evaluated using (i) radiography, (ii) bone length estimation, (iii) histological assessment after hematoxylin and eosin staining, and (iv) measurement of the osteogenic area from histology samples using the Image-Pro Plus 6.0 software (n = 4) [27].Copyright© 2017, Springer Nature.