Peptides for Targeting Chondrogenic Induction and Cartilage Regeneration in Osteoarthritis

Osteoarthritis (OA) is a widespread degenerative joint condition commonly occurring in older adults. Currently, no disease-modifying drugs are available, and safety concerns associated with commonly used traditional medications have been identified. In this review, a significant portion of research in this field is concentrated on cartilage, aiming to discover methods to halt cartilage breakdown or facilitate cartilage repair.

Chondroinductive peptides, synthetically producible, boast superior reproducibility, stability, modifiability, and yield efficiency over natural biomaterials. This review outlines a chondroinductive peptide design, molecular mechanisms, and their application in cartilage tissue engineering and also compares their efficacy in chondrogenesis in vitro and in vivo .

Osteoarthritis (OA) is a debilitating, degenerative condition characterized by the gradual failure of joints. 1 Although the exact cause of OA remains elusive, its hallmark is the breakdown of cartilage and the loss of the crucial extracellular matrix that provides joints with compressive resilience to enable proper function. 2 Currently, disease-modifying osteoarthritis drugs (DMOADs) are lacking, leaving patients with limited options, ranging from pain management in the early stages to surgical joint replacement in advanced ones. This underscores the urgent need for effective treatments for OA. Research efforts have been intensely directed toward understanding cartilage to identify strategies for either halting cartilage degradation or promoting cartilage repair. 3 , 4 Presently, cellular-based approaches to regenerate cartilage focus on increasing the number of cells that can revitalize joints, either by recruiting progenitor cells or enhancing chondrocyte density through techniques such as microfracture or autologous chondrocyte implantation ( Fig. 1 ). This review explores various strategies, with a particular emphasis on peptide-based compounds. These compounds are known for their selective binding to key molecules within the growth factor cytokines and cartilage extracellular matrix (ECM), including type II collagen and aggrecan. We present an updated overview of advances and clinical trials aimed at developing DMOADs that target growth factors and metalloproteinases. Additionally, we explored novel approaches for the delivery and retention of potential OA therapies within the joint environment. The development of peptide carrier systems is increasingly recognized as a promising avenue for mitigating the impact of potential DMOADs on the joint and minimizing the risk of systemic adverse effects. Figure 1. Current cellular-based strategies employed for cartilage regeneration employ various approaches to promote tissue repair and regeneration. Cellular-based strategies for cartilage regeneration aim to increase the population of cells with the ability to initiate or engage in tissue regeneration. These methods typically prioritize either the recruitment of progenitor cells or the enhancement of chondrocyte density. Recruitment strategies involve techniques such as microfracture, aimed at stimulating the migration of progenitor cells from the bone marrow to the injury site, or exosome delivery to attract nearby progenitor cells. Once recruited, these progenitor cells play a crucial role in cartilage repair by modulating inflammatory responses and promoting chondrogenesis through paracrine signaling, potentially undergoing direct differentiation into chondrocytes. Alternatively, chondrocyte enrichment strategies include procedures such as autologous chondrocyte implantation or matrix-assisted autologous chondrocyte implantation, which are often complemented by agents targeting cellular senescence. Moreover, peptide-based approaches for cartilage regeneration are hypothesized to function by promoting chondrogenesis in activated or regenerating states, enhancing cell viability, or inducing the differentiation of chondrocytes derived from MSCs. The carrier strategy incorporates organic compounds, including chitosan, poly (lactic-co-glycolic) acid (PLGA), HA, and polyester amide (PEA), to improve peptide stability and facilitate delivery.

Peptides that drive chondrogenesis are classified into three primary categories: (1) peptides derived from growth factors, (2) peptides derived from cell–cell adhesion molecules, and (3) peptides targeting ECM components, such as type II collagen and aggrecan ( Fig. 3 ). Figure 3 provides an overview of the various chondroinductive peptides, with a particular focus on peptides derived from bone morphogenetic protein (BMP) signaling pathways (casein kinase 2.1 [CK2.1], BMP, B2A2-K-NS (B2A)), which are known to trigger BMP signaling and thereby promote chondrogenesis in mesenchymal stem cells (MSCs) or chondrocytes. It also outlines criteria for assessing their efficacy both in vitro and in vivo . BMP signaling–derived peptides are growth factor–derived peptides and include BMP peptide and B2A. SPPEPS peptide, another notable peptide in this group, is a common sequence found in TGF-β3 and aggrecan. ECM-derived peptides are derived from cell–cell adhesion molecules and ECM components. A notable example is the N-cadherin mimetic peptide, which is derived from the cell–cell adhesion molecule N-cadherin. Peptides derived from ECM components include arginylglycylaspartic acid (RGD) peptide, collagen mimetic peptide (CMP), GFOGER peptide, glycopeptide, and link protein N-terminal peptide (LPP). Figure 3. Schematic of the role of peptides in chondrogenesis. The diagram depicts the category of chondroinductive peptides, detailing their molecular mechanisms and parameters for assessing their in vitro and in vivo efficacies. Specifically, peptides derived from BMP signaling, namely CK2.1 peptide, BMP peptide, and B2A peptide, are shown. These peptides operate by activating BMP signaling, thereby promoting the induction of chondrogenesis in MSCs or chondrocytes. BMP: bone morphogenetic protein; TGF-β: transforming growth factor-β; ECM: extracellular matrix; CMP: collagen mimetic peptide; LPP: link protein N-terminal peptide. Growth Factor-Derived Peptides Bone morphogenetic protein 2 (BMP2) BMP peptide originates from a specific segment (residues 73–92) of the knuckle epitope found in BMP-2. 34 It comprises a sequence of 20 amino acids (KIPKASSVPTELSAISTLYL). BMP peptide is advantageous because, unlike BMP-2, it does not require additional processing, such as disulfide bond formation or complex folding regimens; 17 it also has considerable chondrogenic potential. 18 In a micromass chondrogenesis model with human bone marrow-derived MSC (BMSC) cells, BMP peptide was demonstrated to increase the production of glycosaminoglycan (GAG); this result was comparable to that achieved using BMP-2. However, the efficacy of BMP peptide in enhancing total collagen content was considerably lower than that of BMP-2 at 3 weeks post-incubation. At the initial phase of the experiment (day 3), BMP peptide promoted the expression of Sox9, Aggrecan, and Comp. Moreover, at 4 weeks post-incubation, BMP peptide significantly increased the levels of ALP activity and collagen X, although these increases did not reach the levels observed with BMP-2. Furthermore, BMP peptide was associated with a more evenly distributed matrix molecule presence compared with BMP-2. However, despite these promising in vitro results, a noticeable gap in research regarding the in vivo effects of BMP peptide on cartilage formation exists.

The SPPEPS peptide contains a shared sequence present in two molecules, TGF-β3 and aggrecan. In TGF-β3, the SPPEPS sequence is located in the latency-associated protein region, known as a ligand for various integrins. 38 SPPEPS significantly increased collagen II expression in rat BMSCs compared with the negative control group after 3 days. 20 Moreover, at 7 days, the Kyoto Encyclopedia of Genes and Genomes analysis of the proteomics data revealed that SPPEPS activated insulin signaling pathways through a gene crucial for cartilage development, namely GSK-3β. Additionally, SPPEPS was demonstrated to significantly upregulate the expression of collagen XIα1, a critical contributor of cartilage formation. 20 A gene ontology analysis indicated that SPPEPS upregulated chondrogenesis-related genes such as ENPP1 and CLIC4. 20 When incorporated into pentenoate-functionalized hyaluronic acid (HA) hydrogels alongside RGD, the conjugation of both SPPEPS and RGD significantly enhanced collagen II expression in rat BMSCs by approximately 300 folds compared with the control group. 20 However, data required for the investigation of the in vivo efficacy of SPPEPS remain unavailable.

The GFOGER peptide sequence serves as a recognition site for integrin α2β1, a primary integrin collagen receptor situated within residues 502–507 of the collagen I α1(I) chain. 44 To create GFOGER-modified hydrogels, the collagen mimetic peptide containing GFOGER, with the sequence (GPO)4GFOGER(GPO)4GCG(CMP), is chemically integrated into PEG hydrogels through Michael addition chemistry, thereby facilitating the functionalization of PEG. 28 , 45 Michael addition chemistry, which does not require catalysts or initiators, is employed to form hydrogels from aqueous solutions in the presence of cells, circumventing issues associated with UV exposure and toxic initiators in the photopolymerization process. 28 , 46 Human MSCs (hMSCs) within the GFOGER-functionalized hydrogels exhibited a highly spread morphology, in contrast to the star-like morphology observed in RGD-functionalized hydrogels. Cell proliferation was greater in GFOGER-functionalized hydrogels than in RGD-functionalized hydrogels. Moreover, compared with RGD-functionalized hydrogels, GFOGER-modified hydrogels exhibited significantly higher mRNA expressions of collagen II and aggrecan and higher GAG content in hMSCs. 45 These findings suggest that the GFOGER peptide enhanced cell spreading and proliferation in PEG hydrogels, providing a superior chondrogenic microenvironment compared with the RGD peptide.

LPP, also known as link-N, is a 16-amino acid peptide (DHLSDNYTLDHDRAIH) derived from the cleavage of link protein. 50 , 51 Both the endogenous LPP and its biochemically synthesized counterpart have been demonstrated to be capable of stimulating the synthesis of aggrecan and collagen II in cartilage stem/progenitor cells 52 and intervertebral disk cells. 29 Wang et al . 53 conjugated LPP to RADA16 29 to create a functionalized nanofiber hydrogel scaffold known as LN-NS. 54 LN-NS was found to significantly enhance the adhesion but not the proliferation of BMSCs. Moreover, compared with the RADA16 hydrogel, LN-NS was found to markedly upregulate the expression of chondrocyte-related genes in BMSCs, including collagen II and aggrecan. 54 In primary chondrocytes, LPP selectively binds to BMPR-II, forming a direct peptide–protein association, but does not bind to BMPR-I. This interaction triggers the production of endogenous BMPs, such as BMP-4 and BMP-7. 55 The newly synthesized BMP-7, but not BMP-4, activates p-Smad1/5, subsequently inducing the expression of the chondrocyte-specific transcription factor Sox9. This activation leads to the downstream expression of aggrecan and collagen II. By contrast, inhibitors of PI3K/AKT (LY294002), p38 MAPK (SB203580), or ERK1/2 (U0126) do not hinder the LPP-induced production of aggrecan, collagen II, and Sox9. 55 Levels of Runx2 and collagen X remain unaffected by LPP, indicating that LPP does not influence osteogenic induction. At the protein level, LPP has been demonstrated to stimulate the expression of Sox9, aggrecan, and collagen II, with significant increases observed in Sox9 and aggrecan levels and in collagen II expression. 56 However, no in vivo study has validated its efficacy.

Aggrecan, an abundant ECM molecule in cartilages, is characterized by a high fixed charge density attributed to numerous chondroitin and keratan sulfate moieties. This distinctive feature has been leveraged for cartilage targeting through electrostatic interaction strategies to enhance the binding and retention of positively charged molecules within the cartilage ECM. Peptides such as RRRR(AARRR)3R 61 and proteins such as avidin 62 are among the cationic carriers assessed for cartilage delivery. Notably, avidin-conjugated dexamethasone demonstrated superior efficacy in inhibiting IL-1-driven aggrecan breakdown in cartilage explant cultures compared with soluble dexamethasone, 63 underscoring the potential of this approach. Furthermore, heparin-binding domains in growth factors, such as FGF18, exhibit cationic properties at neutral pH, rendering them suitable for cartilage delivery. For instance, the heparin-binding domain of heparin-binding epidermal growth factor (HB-EGF) has been employed to enhance the retention of IGF-1 in cartilage in vivo , resulting in increased therapeutic efficacy in a rat medial meniscal tear model of OA. 64 Notably, cationic delivery strategies require careful design to support weak, reversible interactions with the cartilage matrix, because an excessively positive charge may favor tight binding, potentially limiting penetrability.

Peptides are susceptible to enzymatic degradation and exhibit low absorption rates, necessitating the exploration of alternative carrier delivery systems. The goal is to preserve the structural integrity of peptides while enhancing their absorption and bioavailability. Additionally, the closed structure of the knee joint and the absence of blood vessels in articular cartilage pose challenges for drugs to accumulate in the joint through the bloodstream. This limitation may lead to reduced efficacy and increased systemic side effects. Conversely, IA injection delivery offers notable advantages, including minimal systemic side effects, rapid achievement of therapeutic concentrations, and rapid pain relief. Consequently, IA injection has gained widespread acceptance as a preferred method of drug delivery in clinical practice. However, repeated IA injections may result in temporary hyperglycemia, skin redness, fever, popular-like changes, local infections, skin atrophy, and periarticular calcification. Therefore, enhancing peptide delivery systems becomes crucial in overcoming constraints associated with conventional injectable dosage forms, which involve repetitive dosing, lack long-term effects, and induce severe side effects. Delivering peptides to avascular tissues with negative charges, such as cartilage, presents a considerable challenge. The continuous turnover of synovial fluid leads to a short residence time for the administered peptides in the joint space, whereas the dense, negatively charged cartilage matrix impedes their diffusive transport. Consequently, peptides encounter difficulties in reaching their cellular and matrix targets in adequate doses, leading to an inability to elicit meaningful biological responses and contributing to unsuccessful clinical trials. Among the different drug delivery systems (DDSs), polymeric nanoparticles, mesoporous silica nanoparticles (MSNs), and liposomes are frequently employed. MSNs stand out as competitive carriers for drug loading due to their customizable pore sizes and significant pore volumes. 68 These features make them suitable for encapsulating a range of pharmaceutical drugs, such as DOX, 69 TPT, 70 and CPT, 71 as well as fluorescent dyes, enabling controlled drug release. Moreover, the extensive surface functionalization capabilities of MSNs allow them to interact with a wide array of agents, including macrocyclic compounds, polymers, dendrimers, biomacromolecules, other nanoparticles, and even therapeutic drugs. 72 , 73 In addition, liposomes are nanoparticles capable of encapsulating both lipid-soluble and water-soluble drugs. This encapsulation safeguards the drug from rapid degradation and reduces its toxicity by limiting its availability in the systemic circulation. Peptide-functionalized liposomes tagged with imaging agents can efficiently and selectively deliver diagnostic agents to targeted sites. 74 For diagnostic applications, methods such as targeting specific receptors with peptide liposomes, utilizing irradiation-mediated diagnostic imaging with peptide-targeting liposomes, and employing peptide-conjugated theranostic liposomes that combine therapeutic and diagnostic functions can effectively detect cancers at various stages. 75 , 76 Additionally, some of these techniques hold potential for developing personalized medicines. Thus, an overview of sustained-release DDSs and permeable DDSs, with a particular focus on polymeric nanoparticles used for IA injections. Chitosan Chitosan (CS), derived from the natural polysaccharide chitin by the removal of a portion of the acetyl group, possesses favorable attributes such as biodegradability, biocompatibility, and nontoxicity. Chitosan finds diverse applications in medical fibers, medical dressings, and peptide slow-release materials. 77 Chitosan nanoparticles have gained recognition as valuable tools for peptide delivery due to their ease of preparation, ability to encapsulate different macromolecules and drugs, and capacity to exert effects on various tissues or organs. Chitosan microparticles (CMs) have been demonstrated to be useful in delivering certain polypeptide drugs. 78 In a study by Chen et al ., dextran sulfate-CMs were developed for the efficient delivery of rhBMP-2. These CMs had a particle size of approximately 250 nm and sustained the release of rhBMP-2 for approximately 20 days. 79 Additionally, HA hydrogel has served as a carrier to enhance CS treatment. 80 HA can be synthesized at varying methacrylic acid levels compared with common hydrogels and allows for the production of hydrogels with adjustable physical properties, including degradation, stiffness, and pore structure, making it promising for applications in tissue engineering. Studies have reported that CMs loaded with cordycepin and delivered by HA hydrogel could stimulate autophagy in the treatment of OA, 81 with experimental results indicating continuous drug release for up to 3 days. Furthermore, hyaluronic acid methac

HA, composed of a disaccharide unit of D-glucuronic acid and N-acetylglucosamine, is commonly used in the construction of drug/peptide delivery systems. 84 It possesses advantageous qualities, including biocompatibility; degradability; and noninflammatory, nontoxic, and nonimmunogenic properties. Therefore, HA is a promising material for sustained-release DDS with dual treatment and peptide-loading characteristics. However, exogenous HA is susceptible to degradation in the joint cavity, with a high clearance rate. Consequently, researchers often modify or protect HA to prolong its retention time in joint applications. One such approach involves modifying HA with the heat-sensitive poly(N-isopropyl acrylamide) (pNiPAM) polymer, which allows HA to spontaneously form nanoparticles at body temperature. This modification enhances resistance to hyaluronidase degradation, effectively extending the residence time at the injection site. 85 Over a 21-day monitoring period, heat-sensitive particles were observed to persist in the joint cavity, in contrast to free HA, which began to diffuse just 1 h after injection. These nanoparticles effectively protected the cartilage, reduced the levels of proinflammatory cytokines, and maintained epiphyseal thickness. The introduction of sulfuric acid groups to modify HA provides resistance against hyaluronidase decomposition and imparts a negative charge to HA. This modification holds significance in binding positively charged residues of proteins, thereby enhancing the capacity of the modified HA to carry peptide drugs. 86 Apart from modification, using HA to create a distinctive three-dimensional structure presents another potential solution. Consequently, HA-based DDSs often exhibit excellent characteristics, highlighting the potential of HA application in peptide delivery. These attributes include ease of modification, sustained release, active targeting, and low side effects. Some peptides are capable of self-assembling into hydrogels for tissue engineering. These peptides include RADA16 (AcN-[RADA]4-COHN2), KLD-12 (AcN-KLDLKLDLKLDL-CNH2), PS-b-PEO-Ada, palmitoyl-V3A3K3–Am, and HSNGLPL. 87 - 89 These self-assembled peptides undergo gelation through noncovalent self-assembly mechanisms, enabling the incorporation of bioactive groups that replicate protein functions. 87 Although these self-assembling peptides primarily function as scaffolds, other peptides have the ability to form thermosensitive hydrogels tailored for cartilage tissue engineering. Polyalanine (PA), poly(alanine-co-phenylalanine), and poly(alanine-co-leucine) conjugated to PEG or poloxamer are some examples of thermosensitive copolymers. The gelation process is facilitated by strong hydrogen bonds or ionic interactions between peptide chains. 90 Notable peptide-based thermosensitive hydrogels include PEG-b-PA, graphene (GO)/PEG-b-PA, reduced GO/PEG-b-PA, PEG-b-PA-b-poly(l-aspartate) (PD), poly(l-alanine-co-l-phenylalanine) (PAF)-b-PEG-b-PAF, and PA-b-poloxamer (PLX)-b-PA. 91 , 92 Liu et al . 91 demonstrated that BMSCs within PAF-b-PEG-b-PAF produced greater amounts of GAGs and collagen II and less amounts of collagen I than PA-PEG-PA and control after a 12-week injection into rabbit cartilage defects in rats. Peptide-based thermosensitive hydrogels have been extensively reviewed elsewhere. 92

Peptide development is an important yet challenging endeavor, and many researchers have focused on inhibiting metalloproteinase-mediated cartilage degradation and promoting cartilage repair. Notably, several promising therapies have advanced to clinical trials, underscoring the success of fundamental research and the identification of robust targets for drug development. Strategically guiding these peptides to the cartilage presents a promising approach to overcome challenges in drug delivery for OA. Leveraging the cartilage matrix as a drug reservoir while minimizing potential systemic toxicity holds particular significance in addressing the chronic nature of OA and its high prevalence of comorbidities. However, despite promising in vitro results demonstrating the successful stimulation of chondrogenic factors during MSC chondrogenesis, in vivo evidence fully supporting the efficacy of peptides in cartilage repair for OA is currently insufficient. Challenges remain, such as the limited lifespan of peptides within the joints and uncertainties regarding their efficiency in enhancing recovery or regeneration. Therefore, as a pragmatic approach, combining chondrogenic induction peptides with organic compounds to create a chondrogenic cocktail treatment may be beneficial. This combination treatment may enhance the modulation of the inflammatory environment and promote the formation of new cartilage in the context of OA. In summary, notable advancements have been made in the development of DDSs for OA treatment. These advancements include improved capabilities for continuous peptide delivery to alleviate OA symptoms and slow disease progression. Nevertheless, DDSs with effective penetration are essential for the development of chondrocyte-targeting drugs that address the underlying causes of OA. In conclusion, IA DDSs have the potential to enhance the penetration of drugs into the cartilage extracellular matrix, facilitating the sustained release of the potential peptides in vivo .