Peptides for Targeting Chondrogenic Induction and Cartilage Regeneration in Osteoarthritis

Abstract

Objects

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.

Methods

Researchers have mainly investigated the cartilage, seeking methods to promote its repair. This review focuses on peptide-based molecules known for their ability to selectively bind to growth factor cytokines and components of the cartilage extracellular matrix.

Results

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.

Conclusions

In this paper, we will summarize the application of peptides engineered to regenerate cartilage by acting as scaffolds, functional molecules, or both and discuss additional possibilities for peptides. This review article provides an overview of our current understanding of chondroinductive peptides for treating OA-affected cartilage and explores the delivery systems used for regeneration. These advancements may hold promise for enhancing or even replacing current treatment methodologies.

Introduction

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 in Cartilage Regeneration

Peptides, which are short chains of amino acids or small proteins naturally synthesized in our bodies, are crucial for a wide range of biological functions. Over the past decade, peptides have been widely applied in drug development. ⁵ They mimic protein functions but are simpler and cheaper to produce. Moreover, peptides have the ability to target specific “flat pockets” on molecules, areas that are considered unreachable by conventional small-molecule drugs. ⁶ Their synthesis is straightforward, and their size, functional groups, and biological activities can be easily modified, making peptides a promising avenue for drug development. Several peptides have demonstrated efficacy in alleviating pain associated with OA and musculoskeletal injuries and promoting tissue healing. Moreover, they have been reported to promote cartilage regeneration in OA and play a role in harmful processes such as osteophyte formation and inflammation, which further exacerbate cartilage degradation (Fig. 2). ⁷ Key peptides that have been recognized for their benefits to bone and joint health include BPC-157, ⁸ thymosin beta-4 (TB 500), ⁹ AOD 9604 ¹⁰ and pentosan polysulfate sodium (PPS, Elmiron), ¹¹ with administration methods varying from oral intake and subcutaneous injection to topical application. Crucially, unlike steroid hormones, which persist in the body for an extended duration, peptides have a short half-life, contributing to a favorable safety profile. Numerous peptides have found applications in treating orthopedic disorders, ¹² with some already being used in clinical trials and preclinical studies (Table 1). For example, parathyroid hormone 1-34 peptides are employed in the treatment of osteoporosis in postmenopausal women. ¹³ In the realm of cartilage tissue engineering, peptides serve as active molecules that facilitate cell adsorption, enrich enrichment motifs, and act as scaffolding. ¹⁴ Therefore, the studies focused on identifying peptides relevant for cartilage regeneration and exploring new prospects for their application in this field. These studies also investigated alternative carrier delivery systems that are designed to (1) overcome existing problems, such as enzymatic degradation and low peptide absorption; (2) maintain structural integrity; and (3) enhance absorption and bioavailability. In addition, the studies addressed methods for optimizing peptide carriers to minimize degradation.^15,16

Figure 2.

Role of peptides in the management of osteoarthritic joints after treatment. Peptides play a crucial role in the treatment of osteoarthritic joints. In a healthy joint, peptides promote cartilage regeneration triggered by mechanical loading, thereby preserving the differentiated chondrocyte phenotype. Conversely, in an osteoarthritic joint, various damaged cartilage phenotypes are observed, accompanied by alterations in chondrocytes. These signals of damage lead to osteophyte formation, chondrocyte hypertrophy, synovial inflammation, increased bone turnover, and the degradation of articular cartilage. Furthermore, the presence of inflammatory cytokines and changes in the cartilage microenvironment exacerbate the propensity of articular chondrocytes toward hypertrophy.

Table 1.

Peptides Currently Used for Treating Cartilage Damage in Patients with OA, Demonstrating in Vivo Efficacy and Inducing Chondrogenesis In Vitro.

Peptide	Study Design	Model	Results	Reference
BPC-157	1. BPC-157 alone 2. Combination of BPC-157 and thymosin beta-4 (TB4)	16 Patients With OA	Multiple types of knee pain were alleviated.	Dönges et al. 7
Thymosin beta-4 (TB 500)	Treatment of primary chondrocytes with thymosin beta-4 peptide	Articular cartilage chondrocyte, in vitro.	TB4 significantly increased the expression and activation of pro-MMP 9.	Lee and Padgett 8
AOD 9604	Weekly injections of 0.6 mL of saline (Group 1), 6 mg of HA (Group 2), 0.25 mg of AOD9604 (Group 3), and 0.25 mg of AOD9604 with 6 mg of HA (Group 4) were administered for 4–7 weeks in the collagenase-induced knee OA rabbit model.	Collagenase-induced Knee OA rabbit model.	Intra-articular AOD9604 injections enhanced cartilage regeneration in the collagenase-induced knee OA rabbit model.	Blain et al. 9
Pentosan polysulfate sodium (PPS, Elmiron®)	Oral administration (10 mg/kg once every 4 days for 5 weeks for two cycles) to individuals with painful knee OA and a history of primary hypercholesterolemia	38 participants, mean age: 62.2 years	PPS exerted promising effects on the improvement of dyslipidemia and alleviation of symptomatic pain relief in patients with knee OA.	Kwon and Park 10
BMP2 peptide	20 amino acids (KIPKASSVPTELS AISTLYL)	A micro mass chondrogene sis model using human BMSCs cells, in vitro	BMP peptide stimulated the expression of SOX9, aggrecan, COMP and increased GAG content.	Liu et al. 15
B2A	BMP receptor-targeting domain (AISMLYLDENEK VVL) sourced from amino acid residues 91–105 of mature human BMP-2	1. Microma ss Chondrog enesis model of C3H10T 1/2 cells, in vitro 2. Mono-iodoacetate (MIA)-induced OA animal model	In a rat model of OA induced by MIA, treatment with B2A significantly enhanced levels of cartilage GAGs and increased the density of cartilaginous cells.	Previous works17,18
SPPEPS	The SPPEPS peptide represents a shared sequence found in two molecules, TGF-β3 and aggrecan.	Rat BMSCs, in vitro	SPPEPS upregulates chondrogenesis-related genes such as ENPP1 and CLIC4.	Lin et al. 19
RGD	Peptides such as GGGGRGDY, GCGYGRGDSPG, GRGDSP, and GGGGRGDSY	Human BMSCs, in vitro	Incorporation of RGD markedly boosted chondrogenesis, as evidenced by the increased mRNA expression of Sox9, collagen II, and aggrecan, along with increased levels of aggrecan and collagen II.	Previous works20 -25
Collagen mimetic peptide (CMP)	Specific peptide sequence —(P[ xyl]PG) x —	MSCs treated with CMP/PEOD A hydrogels for 3 weeks, in vitro	MSCs in CMP/PEODA hydrogels significantly elevated the levels of GAGs, total collagen, aggrecan, and collagen II.	Previous works26,27
GFOGER peptide	Peptide sequence (GPO)4GFOGER(GPO)4GCG(CMP)	MSCs treated with GFOGER-modified hydrogels, in vitro	GFOGER-modified hydrogels significantly increased the mRNA levels of collagen II and aggrecan and GAG content in hMSCs at 7 days and 21 days.	Liu et al. 28
Link protein N-terminal peptide (LPP)	16-amino acid peptide (DHLSDNYTLDHDRAIH)	Cartilage stem/progeny tor cells, in vitro	LPP stimulated the expression of Sox9, aggrecan, and collagen II. Similarly, at the protein level, it increased the levels of Sox9, aggrecan, and collagen II.	Ma et al. 29
Type II collagen-binding peptide	Peptide sequence (WYRGRL)	Cartilages dissected from both tibial plateaus, in vivo	Collagen-binding avimers demonstrated remarkable retention in rat knees for up to a month.	Formica et al. 30
Aggrecan-binding peptide	Peptide sequence (RRRR[AARRR]3R)	Cartilage explant cultures, in vitro Avidin	Avidin and cationic carriers, such as RRRR(AARRR)3R, have been evaluated for their potential in delivering substances to cartilage. Avidin-conjugated dexamethasone demonstrated higher effectiveness in preventing IL-1-induced aggrecan breakdown in cartilage explant cultures than soluble dexamethasone.	Previous works31 -33

Classification of Chondrogenic Peptides

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.

B2A2-K-NS (B2A) peptide

B2A peptide is an engineered molecule, chemically synthesized to include two primary functional domains: one that targets BMP receptors and another that binds to heparin.^19,35 These two domains are linked together by a hydrophobic spacer (KK-(NH[CH2]₅CO))3. ³⁵ The segment of BMP that interacts with BMP receptors (AISMLYLDENEKVVL) is derived from amino acid residues 91–105 found in the mature human BMP-2 protein. ³⁶ Lin et al. demonstrated that B2A alone significantly enhanced ERK1/2 activation and inhibited the phosphorylation of Smad1/5/8 in C2C12 cells. However, the addition of BMP-2 suppresses p-ERK activation and significantly increases p-Smad1/5/8 levels. Crucially, B2A exhibits strong specificity toward BMPs, with no response to other growth factors such as fibroblast growth factor-2 (FGF-2), TGF-β1, and vascular endothelial growth factor. In clinical scenarios, B2A-coated ceramic granules have been employed as a substitute for bone in the treatment of end-stage hindfoot arthritis.

B2A has also been demonstrated to enhance chondrogenesis. In the micromass chondrogenesis model of C3H10T1/2 cells, microarray analysis revealed that B2A significantly increased mRNA expression of Sox9, collagen II, Fgf1, Fgfr1, Fgfr2, Twist1, and PDGF AA. ³⁷ Consistent with this finding, B2A was demonstrated to enhance the protein expression of collagen II and PDGF AA in murine C3H10T1/2 cells. Furthermore, in a rat model of OA induced by mono-iodoacetate (MIA), B2A treatment significantly enhanced the quality of cartilage, as evidenced by increased GAG production and a higher density of cartilaginous cells. ³⁷

TGF-β3-derived peptides

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.

Peptides Derived from Cell–Cell Adhesion Molecules

Integrins primarily facilitate cell–ECM adhesion and serve as receptors for ECM proteins. Each integrin is composed of one α and one β subunit, with a total of at least 16 α subunits and 8 β subunits available. Various combinations of these subunits can form integrin complexes. Both subunits comprise three domains: an extracellular domain, a cytoplasmic region, and a transmembrane domain.^21,22 RGD and GFOGER, derived from specific ECM proteins, rely on integrins to induce chondrogenic differentiation.

Arginylglycylaspartic acid (RGD)

RGD, recognized as a typical cell adhesion motif in various adherent ECM, blood, and cell surface proteins, ²³ binds to integrins on the cell membrane, facilitating cell adhesion, spreading, and other cellular activities. The minimal sequence of RGD is often combined with different amino acid linkers to form RGD peptides, such as GGGGRGDY,^24,25 GCGYGRGDSPG,^39,40 GRGDSP, ⁴¹ and GGGGRGDSY. ⁴² The incorporation of RGD has been demonstrated to significantly enhance chondrogenesis, as evidenced by the increased mRNA expressions of Sox9, collagen II, and aggrecan as well as elevated levels of aggrecan and collagen II expression. In alginate, RGD did not directly enhance chondrogenic differentiation; instead, it substantially increased levels of TGF-β1-induced key chondrogenic signaling molecules, such as p-Smad2/3 and p-ERK1/2, thereby promoting chondrogenesis. ²⁴

Conversely, a single integrin heterodimer has the ability to identify several ECM proteins, and a specific ECM ligand may be identified by more than one integrin. ²⁶ Various integrin receptors, including αvβ3 and α5β1, demonstrated the ability to directly recognize and bind to the RGD motif. ²⁷ Integrin receptors differ in their effects; for example, as demonstrated in a previous study, αvβ3 plays distinct roles in adhesion and spreading, whereas α5β1 is crucial for differentiation. ⁴³ The use of the anti-αvβ3 antibody significantly downregulated cell spreading but the use of the anti-β1 antibody and anti-α5 antibody upregulated cell spreading.^41,42 These findings suggest a competitive relationship among receptors for RGD ligands, with αvβ3 integrin being primarily implicated in cell spreading and cartilage formation. ⁴¹

GFOGER peptide

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.

Peptides Targeting ECM Components

Collagen mimetic peptide

CMP, characterized by a specific amino acid sequence—(P(hydroxyl)PG) x—exhibits a strong affinity for both native and gelatinized collagen I under controlled thermal conditions. CMP has a triple helix conformation that closely mimics the natural protein structure of native collagens. ⁴⁷ This triple helix structure, identified as (P(hydroxyl)PG)7, can be further stabilized by the inclusion of a tyrosine (T), thereby increasing its melting temperature. ⁴⁷ When ((P(hydroxyl)PG)7-T) is conjugated to poly(ethylene oxide) diacrylate (PEODA) hydrogel, it alters the bioinertia and non-cell-adhesive properties of PEODA, creating a favorable bioactive microenvironment that fosters the efficient chondrogenic differentiation of MSCs.^48,49 After a 3-week culture period, an immunostaining analysis was performed, which revealed that the expression of collagen II in MSCs in CMP/PEODA hydrogels significantly increased and that of collagen X significantly decreased compared with their levels in PEODA.^48,49 Furthermore, MSCs in CMP/PEODA hydrogels demonstrated significantly elevated levels of GAGs, total collagen, aggrecan, and collagen II compared with MSCs in PEODA hydrogels.^48,49 However, the current data are insufficient to conclusively determine the in vivo efficacy of CMP.

Link protein N-terminal peptide (LPP)

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.

Type II collagen-binding peptide

Type II collagen, predominantly found in adult cartilage with a low turnover rate, is a promising target for potential DMOADs. Rothenfluh et al. employed a phage display of peptide libraries to identify a peptide (WYRGRL) that selectively binds to type II collagen. Notably, the WYRGRL-targeted fluorescent nanoparticles exhibited a 72-fold higher signal after 48 h compared with nanoparticles comprising a scrambled peptide. ⁵⁷ Subsequently, the WYRGRL peptide was employed to deliver various cargo to cartilage, including dexamethasone, ³⁰ pepstatin A, ⁵⁸ and an HA-binding peptide.^31,59

A recently devised approach for collagen targeting involves avimers, which are artificial binding proteins engineered for high-affinity binding to specific target molecules. Hulme et al. employed protein A domains from various cell surface receptors as the avimer scaffold to generate avimers with a high affinity for type II collagen through in vitro exon shuffling and phage display techniques. ³² These collagen-binding avimers demonstrated remarkable retention in rat knees for up to a month following intra-articular (IA) injection. When the avimer was fused with IL-1Ra, the resulting construct exhibited the ability to block IL-1 activity in rat knee joints in vivo, even when administered a week before the IL-1 challenge. ³²

Phage display has been employed to generate single-chain variable fragments (scFv) capable of binding to type II collagen modified by reactive oxygen species. These antibodies exhibited selective binding to damaged rheumatoid and osteoarthritic joints, ³³ indicating a targeted approach toward regions of joint damage. While retaining their binding to cartilage, these fragments were able to carry payloads such as an MMP-cleavable form of viral IL-10 ⁶⁰ and soluble TNF receptor II, ³³ enabling the in vivo imaging of murine OA cartilage. However, due to the relatively large size of scFv fragments (≈27 kDa), avimers (≈4 kDa) and peptides (850 Da for WYRGRL) are preferred for constructing targeted DMOADs.

Aggrecan-binding peptide

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.

Glycosaminoglycan (GAG) mimetic peptide

GAGs are crucial components of the extracellular matrix in cartilage tissue, providing essential biological signals to MSCs and chondrocytes for cartilage development and functional regeneration. Among their many roles, sulfated GAGs bind to growth factors, enhancing their activity by facilitating growth factor-receptor interactions. ⁶⁵ The growth factor binding properties of native sulfated GAGs can be integrated into synthetic scaffold matrices through functionalization with specific chemical groups. ⁶⁶ A previous study utilized peptide amphiphile nanofibers functionalized with chemical groups from native GAG molecules, such as sulfonate, carboxylate, and hydroxyl, to induce chondrogenic differentiation in rat MSCs. MSCs cultured on GAG-mimetic peptide nanofibers formed cartilage-like nodules and deposited cartilage-specific matrix components. Notably, the degree of chondrogenic differentiation could be adjusted by varying the sulfonation degree of the nanofiber system. ⁶⁷ The GAG-mimetic peptide nanofiber network described here may offer a customizable, bioactive, and bioinductive platform for stem-cell-based cartilage regeneration studies.

Carriers Applied to Enhance the Stability of Peptides

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 methacrylate (HAMA) hydrogel delayed the progression of surgically induced OA at 4 and 8 weeks compared with the control group of OA mice. ⁸² The sustained-release properties of CS hold significant potential for applications in cartilage engineering when combined with hydrogel. In summary, CS is a versatile candidate for developing sustained-release peptide delivery systems in joint applications.

Poly(Lactic-co-Glycolic) Acid

Poly(lactic-co-glycolic) acid (PLGA) is a degradable and functional organic polymer known for its excellent biocompatibility, superior encapsulation performance, and film-forming capacity. It is extensively employed in pharmaceuticals, medical engineering materials, and the modern chemical industry. The injectable nature, degradability, and sustained-release properties of PLGA make it an ideal choice as a peptide carrier. ⁸³ Currently, numerous studies are leveraging PLGA to transport small-molecule drugs, with the objective of extending their residence time within the joint cavity.

Hyaluronic Acid

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

Polyester Amide

Polyester amide (PEA) is a novel polymer derived from amino acids, aliphatic dicarboxylic acids, and aliphatic diols. PEA is biodegradable and amphiphilic and demonstrates notable stability in phosphate-buffered saline (PBS). Numerous studies have highlighted its efficacy as a robust and safe platform for topical drug and peptide delivery. ⁹³ Furthermore, PEA exhibits sensitivity to serine proteases, commonly found in inflammatory environments, making it suitable for DDS in OA treatment. ⁹⁴

Janssen et al. ⁹⁵ investigated the potential of celecoxib-loaded PEA microspheres as a DDS for OA treatment. Notably, these microspheres were observed to be capable of automatic adjustment: they exhibited enhanced degradation in OA joints, and the loading of celecoxib significantly inhibited this degradation. Inflammatory cells were found to sustain the enzymatic degradation of PEA microspheres, and celecoxib effectively impeded this process. The celecoxib-loaded PEA microspheres, with a particle size ranging from approximately 10 to 100 µm, demonstrated excellent sustained-release and retention characteristics. The study observed that the drug was initially released in a burst on the first day, and this accounted for approximately 15% of the total drug load. Subsequently, celecoxib exhibited sustained and slow release for up to 80 days. Furthermore, the celecoxib-loaded PEA microspheres demonstrated favorable biocompatibility and extended retention in the joint cavity and is thus advantageous for OA treatment.

In another study, PEA microspheres loaded with triamcinolone acetonide (TAA) were prepared. ⁹⁶ These microspheres exhibited a sustained release of TAA in PBS for over 60 days and effectively suppressed inflammation for a minimum of 28 days. Using a near-infrared marker, researchers monitored the in vivo fate of the microspheres following administration into healthy rat joints or mildly collagenase-induced OA joints and revealed a retention time of up to 70 days. These findings indicated that these microspheres served as a safe DDS without inducing foreign body reactions, leading to reduced TAA plasma levels and significant alleviation of synovial inflammation after treatment.

In summary, PEA microspheres exhibit excellent biocompatibility, enabling prolonged retention in the joint cavity. Their ability to respond to inflammation establishes them as autoregulatory drug delivery systems, rendering PEA microspheres ideal for use in the development of sustained-release DDS for OA. Additionally, PEA could be a potential alternative for loading candidate peptides aimed at treating OA.

Conclusions

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.