Use of Acellular Matrices as Scaffolds in Cartilage Regeneration: A Systematic Review

SCOPE OF REVIEW, SIGNIFICANCE, TRANSLATIONAL AND CLINICAL RELEVANCE

Cartilage regeneration remains a significant challenge in the field of regenerative medicine. In recent years, considerable focus has been directed toward acellular matrix (AM)-based approaches for cartilage regeneration. This review aims to present a comprehensive overview of numerous studies, encompassing in vitro combined with in vivo findings, regarding various scaffold types, materials, and cell types used for scientific evaluation. Overall, the relevance of finding an optimal scaffold for cartilage tissue engineering with acellular matrices lies in its potential to provide effective and durable solutions for cartilage repair and regeneration, thereby improving the quality of life for patients with cartilage defects.

AMs as cartilage regeneration scaffolds are particularly relevant in the context of designing a biocompatible and safe cartilage regeneration construct for implantation into the body. Translating this technology to clinical use requires widespread testing to ensure that the matrices do not elicit adverse immune responses or cause toxicity. The overall goal is to regenerate functional cartilage tissue that closely resembles native tissue in terms of structure and mechanical properties. Translational research focuses on optimizing the composition and architecture of AMs to promote cartilage tissue regeneration.

Cartilage regeneration technologies such as the development of AM-based therapeutic options are highly relevant in the context of treating cartilage defects caused by injury, degenerative diseases such as osteoarthritis, or congenital abnormalities. These defects often lead to pain, limited mobility, and impaired joint function. AMs could be used in various surgical procedures, including arthroscopic implantation or open surgical techniques. Translating cartilage tissue engineering with AMs from the laboratory to the clinic involves conducting preclinical studies to demonstrate safety and efficacy in animal models, followed by clinical trials in human patients.

INTRODUCTION

Cartilage regeneration is considered a significant challenge in modern orthopedic and regenerative medicine owing to the limited intrinsic capacity for self-repair. ¹ Injuries or degenerative conditions affecting articular cartilage often lead to pain, impaired joint function, and diminished quality of life for affected patients. Traditional treatment approaches, such as surgical interventions and conservative medical therapy, have shown limited efficacy in promoting long-term cartilage repair and restoration of joint function. Advantages such as histocompatibility and overcoming transplantation and prosthesis issues are described as important factors in exploring novel therapeutic strategies for cartilage regeneration. ^{2 –6}

Recent advances in tissue engineering and regenerative medicine have spurred the development of novel therapeutic strategies aimed at promoting cartilage regeneration. Among these strategies, acellular matrix-based (AM) therapeutics, coupled with (stem) cell-based approaches, seem to be promising for enhancing cartilage repair and regeneration. ^4,7,8 AMs offer a scaffold-like structure that mimics the native extracellular matrix (ECM) of cartilage, providing mechanical support and bioactive assistance necessary for cell adhesion, proliferation, and differentiation. ⁶ Regarding immune compatibility, AM-based therapeutics could have a superior position in comparison with present clinical applications. ⁹

The scaffold materials used differ in their origin. Some scaffolds are processed and produced synthetically by 3D printing or bioreactors. ^{10 –12} A wide range of studies use allogenous scaffolds by decellularizing the target tissue or modifying their structure and seeding cells on them. In vivo studies are being conducted to evaluate their biocompatibility for further steps toward clinical studies. Next to primary chondrocytes, stem cells, with their unique capacity for self-renewal and multilineage differentiation, hold immense potential for cartilage regeneration when seeded on acellular scaffolds. ¹³ Special attention is given to mesenchymal stem cells (MSCs) because of their ability to differentiate into chondrocyte-like cells and thereby contribute to the formation of functional cartilage tissue. ¹⁴ Moreover, their ability to reduce inflammatory reactions by anti-inflammatory cytokines is promising for in vivo approaches. ^14,15 When combined with AMs, cells can be seeded onto or within the scaffold facilitating their retention, proliferation, and differentiation at the site of cartilage defects. ¹⁴

This systematic review aims to critically evaluate the current body of literature covering AM-based approaches in the field of cartilage regeneration. A specific focus has been put on the comparison of the different materials that cells and additives used in the studies. By focusing on in vitro studies, we evaluate characteristics in different methods providing a broad insight into the state of basic research in AM-based cartilage engineering. While categorizing and analyzing the available evidence by providing a table reflecting the study details, we seek to provide insights that may inform clinical practice, guide further research, and ultimately contribute to the overall advancement of therapeutic strategies for cartilage regeneration. In addition, we aim to identify gaps in knowledge, unresolved challenges, and future directions for research and clinical application in the field of cartilage tissue engineering.

MATERIALS AND METHODS

Study selection process

The study selection process was based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement, which is shown in the flow diagram (Fig. 1). To further refine the suitable studies for our review, we decided to exclude the following study characteristics: clinical trials only, reviews, editorials, no cartilage evaluated, in vivo only studies. Articles that had only abstracts available were included in our research process but marked with an asterisk symbol (*) when mentioned in this article. The focus of our systematic review lies on the evaluation of in vitro approaches of cartilage engineering. To include the whole basis of research and evaluate the in vivo practicability, in vitro approaches described in the same articles as in vivo study results were included in the Results and Discussion sections as well.

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Figure 1.

Presenting the study selection process via PRISMA. PRISMA, Preferred Reporting Items for Systematic Reviews and Meta-Analyses.

RESULTS

As seen in the PRISMA flow diagram (Fig. 1), our aim was to provide a broad insight into multiple in vitro approaches of acellular cartilage matrix (ACM)-based cartilage tissue engineering. Thus, we excluded only duplicates, only in vivo studies, and articles that did not fit our search terms. By including abstract-only research articles, we tried to not exclude important information suitable for capturing a whole image of the state of current research. In the end, 88 research articles were identified via PubMed search. Whenever possible full texts were obtained and reviewed. Most of the studies were conducted in vitro before subsequently evaluating the results in vivo. A total of 63.6 percent of studies were in vitro studies with additional in vivo testing. A total of 36.4 percent of studies were only conducted in vitro (Fig. 2). As described in the Methods section, only in vivo studies without new direct-linked relevant findings in vitro were excluded in the screening process.

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Figure 2.

Study types separated by in vitro only and combined in vitro+in vivo studies.

Analysis of studies published revealed a distinct increase in recent years, with some fluctuations observed (Fig. 3). This trend suggests a significant presence of research activity in the field of AM-based cartilage engineering, with notable increase in publications especially in the last decade from 2014 to 2023.

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Figure 3.

Published studies (88) meeting inclusion criteria or this review (1999 until the end of 2023).

In Table 1, various scaffold materials and scaffold–cell combinations are described. We only included studies with full text, to provide profound information by being able to scan the full texts of research studies. Moreover, some studies still had crossovers, which we have not mentioned in the table. Scaffold fabrication processes for assessing their potential in cartilage regeneration are predominantly conducted via individual methods by different research groups. We categorized the scaffolds into distinct groups to provide an overview of diverse strategies for ACM-based cartilage engineering. The Hydrogel+X group depicts approaches that amalgamate various hydrogels with ACM, or with collagen products or synthetic polymers such as polyvinyl alcohol, polycaprolactone (PCL), and polylactic acid. ^{10,16 –22} On the contrary, decellularized cartilage matrices (DCMs) without the use of hydrogels were combined with different synthetic polymers as observed in group DCM+X (Fig. 4). In certain studies, microsphere-based scaffolds were evaluated. These encompass synthetic polymers (predominantly polylactic-co-glycolic acid [PLGA]) and ACM from different sources. In addition, some studies evaluated the effect of so-called second delivery carriers such as graphene oxide (GO) and microsphere-based delivery of growth factors. ^23,24 Chondrogenic growth factors such as insulin-like growth factor 1 (IGF-1) or transforming growth factor ß3 (TGF ß3) are encapsulated within microspheres to assess the efficacy of a target-specific application in scaffolds. ^{24 –26} Besides, the incorporation of GO in scaffolds is often combined with the utilization of growth factors. ^23,27

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Figure 4.

(a) Number of studies using different cell types related to scaffold groups as described above. (b) Distribution of cell types across all studies in percent.

A substantial number of studies assessed DCM-only scaffolds. The process for constructing and evaluating this scaffold-type adheres to specific protocols: researchers procure cartilage matrices (CMs) from various animals and anatomical sites (e.g., meniscus, ear, distal femur). ^{28 –30} * Subsequently, the CMs undergo decellularization and are seeded with MSCs, articular chondrocytes (ACs), or adipose derived stem cells (ADSCs). ^{31 –34} Simultaneous usage of MSCs and ACs was observed in some studies. ^{34 –36} Apart from ACM, the functionality of acellular dermal matrices (ADMs) of animal origin is evaluated in some studies for cartilage engineering. The procedural aspects in these studies mirror those evaluating ACM. ^37,38 Furthermore, a study by Wang et al. compared porcine ACM and ADM by scrutinizing the growth dynamics of chondrocytes within the matrices. Findings suggest the advantages of ADM over ACM in terms of chondrocyte distribution. ³⁹

In recent years, novel strategies have emerged to combine diverse tissue types, such as cartilage and bone, aimed at enhancing osteoarthritic regeneration. The basic framework of these biphasic scaffolds is designed to facilitate chondrogenic growth on one side and osteogenic growth on the other side of the scaffold. In vitro approaches combine synthetic materials such as PCL with AMs derived from cartilage and/or bone, along with various hydrogels to construct a scaffold accommodating both chondrogenic and bony phases. ^{40 –44} Findings from these studies implicate successful outcomes both in vitro and in vivo regarding the integration of produced osteochondral grafts into bone, although challenges such as immune system compatibility within in vivo models persist. ^40,41,44

Another approach to build different cell layers within a scaffold involves using cell sheet technology. Studies try to construct cell-ACM-cell models by interlinking them together and stacking them on top of each other. ^{45 –47}

By comparing different commercial matrix products in various configurations, Voss et al. sought to find the best match for cartilage engineering among commercially available matrices. Most promising results in chondrogenic differentiation were yielded by the collagen scaffold (Mucograft), characterized by the highest concentrations of type-2 collagen and aggrecan, as well as a homogenous cell distribution. ⁴⁸

Within our research, we evaluated the kind and frequency of the different cell types used for seeding onto specific scaffold types (Fig. 4). MSCs and chondrocytes were the predominant cell types utilized for assessing the biocompatibility and chondrogenic competence of scaffolds used. However, adipose-derived stem cells (ADSCs) were also commonly used as cell lines, particularly in ACM-only scaffolds. Within composite and biphasic scaffolds, MSCs emerged as the predominant cell type, whereas in hydrogel and ACM scaffolds, primary chondrocytes have been used as commonly to evaluate cell growth. As seen in Table 1, cells used for bioevaluation have several origins, either coming from human donors or animals. Rabbit, cow, and pig seem to be the most common donor animals for cell lines in ACM-based approaches; human cells were used in about a quarter of all the studies analyzed.

Articular cartilage regeneration is the main focus in research of cartilage engineering and reconstruction. Figure 5 exhibits these findings. However, there are also efforts directed toward regenerating other types of cartilage tissues and related structures. Research is being conducted on regenerating auricular cartilage. This type of cartilage is found in the external ear and has distinct properties compared with articular cartilage. ^{9,20,22,31,33,71} * Some studies focus on regenerating the nucleus pulposus, a component of intervertebral discs in the spine. ^62,72,73 The meniscus of the knee is also being targeted for regeneration. ⁵⁶ *^,11,34,74 Both meniscus and nucleus pulposus are referred to as fibrocartilages. ⁷⁵ Besides, there is also research being conducted on cartilage tissues in the larynx, nasal septum, and trachea. Cell-seeded ACM models of animals are being utilized for evaluation and potential regeneration. ^{52,55,63 –65,76} **

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Figure 5.

Distribution of cartilage types and entities in studies investigating their regeneration.

Overall, while articular cartilage regeneration is the major focus, research efforts are also diversified to address the regeneration of various other cartilage tissues and related structures.

DISCUSSION

The primary objective of this review lies in the evaluation of various approaches on ACM-based cartilage regeneration. We presented a comprehensive list of scaffold types along with their distinct characteristics and methodologies for implementing them in studies. Beyond that we provide an overview of cell types utilized for biocompatibility assessment, as well as the origins of biological scaffolds comprising ACM and ADM sourced from the various animals used. Through comprehensive analysis of the studies, we illustrate diverse approaches and recent trends in cartilage engineering within the scope of our investigation.

In general, scaffolds, primarily produced individually, undergo analysis after decellularization and in vitro processing through cell seeding, using various methods. Collagen content is assessed by analyzing various chondrogenic genes, as evaluated in prior studies. ⁷⁷ Genes crucial for evaluating chondrogenic growth success include SOX9, ACAN (aggrecan), COL1A1, COL2A1, COMP, and others. ⁷⁷ Upregulation of these genes generally indicates proliferation and tissue differentiation into cartilage. Scanning electron microscopy images are commonly captured to gain insights into the morphology of the produced scaffolds. ^4,78 Evaluation of mechanical properties is also a common approach before scaffold bioimplementation. This includes testing swelling ratios and compression modulus as described exemplary by Kim et al., as well as measuring the pore sizes and water uptake ratios. ¹⁹ Skaluure et al. identified osmolarity as an indicator for collagen synthesis in their scaffold. ⁷⁹ Lower osmolarities supported midterm synthesis, while higher osmolarities showed short-term benefits. ⁷⁹ However, the lack of sufficient data prevents the establishment of specific osmolarities as standards for scaffold production. Another important factor for successful clinical applications is the long-term mechanical durability of AM. ⁸⁰ Unfortunately, there is not much sufficient research found in the long-term analysis of mechanical properties, since most studies in vitro and, more importantly, in vivo only show short-term results after several weeks of evaluation. ⁸⁰

Analysis of scaffold pore size leads to heterogenous findings because of a substantial number of studies evaluating the impact of pores on the growth and differentiation potential. ⁸¹ Zhang et al. suggest that pore sizes ranging from 150 to 250 μm could adequately support cartilage growth in vitro within a synthetic PCL scaffold. ⁷ On the contrary, their 2016 in vivo study did not demonstrate evidence of beneficial growth in certain pore-sized scaffolds. ⁷⁸ Nava et al. show in a literature review that scaffold pore sizes can generally influence chondrogenic growth in scaffolds. ⁸¹ A common challenge remains in the individualities in scaffold production. This complicated the implementation of homogenous techniques across a wider range of in vitro ACM-based approaches.

The use of growth factors in cartilage engineering is of great importance in various approaches, as demonstrated in several of the studies evaluated. The most commonly used growth factors in cartilage tissue engineering are TGF ß1/3, fibroblast growth factor-2, bone morphogenetic protein 2/7, and IGF-1, extensively described by Fortier et al. ⁷⁷ Positive impacts of growth factors have been observed in several of our evaluated studies, with some using secondary delivery carriers such as GO and microspheres for growth factor delivery. ^{23 –27} In addition, Zhao et al. identified kartogenin as a growth factor in an ACM/PLGA scaffold, demonstrating a similar chondrogenic differentiation capacity as TGF ß3. ²⁵ This finding holds potential for further research, as underlined by the recent studies of Chen et al., highlighting the potential of kartogenin in cartilage regeneration therapeutics. ⁸² The application of GO has been reported to have positive impacts on scaffold structure as well as growth factor distribution. ^23,27 Still, it is a niche product in cartilage tissue engineering and must undergo further evaluation in future research.

The incorporation of microspheres within scaffolds presents an intriguing approach in cartilage regeneration, offering the ability to control the distribution of growth factors or other chondrogenic-enhancing molecules through controlled release and shaping of the microspheres as required prior implantation. ⁸³ Higher porosities have been associated in using microspheres, as well as enhanced cell attachment. ^24,25 Some challenges such as a lack of sufficient chondrogenic induction need to be addressed. ²⁴ Furthermore, limitations in predicting outcomes with the use of microspheres are described in the literature. ⁸³

In vitro approaches in evaluated studies demonstrate the beneficial use of MSC-derived exosomes, aligning with recent literature highlighting exosomes’ significant role in cartilage regeneration. ⁸⁴ Specifically, advantages such as cell protection through inhibition of inflammation and improved cell interaction have been observed in different studies using MSC-derived exosomes. ^30,57

ACMs are extensively used in cartilage engineering investigations because of their inductive environment for chondrocyte growth and cartilage formation by retaining the cartilage-specific ECM structure and chondrogenic-functional proteins. ^39,74 However, ADMs are currently seen as an alternative for ACMs in cartilage tissue engineering, demonstrating the capability to generate proper cartilage tissue. ^37,38,66,67 Recent studies have highlighted advantages such as the increased availability of ADM sources and reduced costs, underlining the significance of ADM research for future applications. ^{37 –39} Results of Wang et al. even suggest beneficial utilization of ADM over ACM, as evidenced by a more homogenous chondrocyte distribution in direct comparison of ADM with ACM. ³⁹ Nevertheless, ACM remains the most studied type in AM research, prompting further exploration of ADM to uncover new possibilities in cartilage regeneration, regarding its practical advantages.

Biphasic scaffolding represents a highly interesting approach because of its capacity to simultaneously enhance osteogenic and chondrogenic regeneration within a single product. Studies imply advantages in addressing challenges to integrate cartilage into the subchondral bone by using a biphasic scaffold construct. ^40,41,43,44 Remarkably, there is a major use of MSC and ADSC for evaluating these scaffolds, in contrast to the balanced use of chondrocytes and pluripotent cells observed in other scaffold compounds (Fig. 5). This preference may be reasoned by the requirement for tissue growth into bone and cartilage, where pluripotent stem cells are indispensable. The combination of ACM and bony phase, for example, hydrogel and MSC/ADSC, has been demonstrated successfully in several studies, including in vivo follow-ups. ⁴³ *^,41 However, challenges such as biocompatibility in larger defects and animal models are apparent in recent and future studies. ⁴⁰ Various in vitro approaches are being pursued to identify the optimal construct for integrating scaffolds into the biological environment. The significant potential of biphasic scaffolding must be further explored in cartilage regeneration research, to overcome issues in both bone and cartilage regeneration. New techniques to archive excellent chondrogenic and osteogenic growth, as well as proof of biocompatibility through both in vitro and in vivo studies, are required.

A relatively underexplored area within AM-based cartilage regeneration lies in the utilization of commercially available ACM or ADM. Within our research, only a limited number of approaches using commercially available products were identified. ^17,48,49 The advantages of using commercial products include their availability and consistent quality, along with demonstrated biocompatibility in clinical studies within other domains of tissue regeneration such as soft tissue repair and bone regeneration. ^85,86 Voss et al. identified the bilayered collagen scaffold of Mucograft as a promising option for cartilage repair, regarding results and chondrogenic markers, as well as a homogenous distribution of cells. ⁴⁸ Further research evaluating the commercial ADM and ACM should be pursued to assess the feasibility of using these readily available matrices for cartilage tissue engineering purposes.

The utilization of matching cells emerges as a significant factor for generating an optimal bioactivated scaffold for in vivo applications. ⁸⁷ Thus, MSCs are highly regarded for their pronounced capabilities in differentiation, immune compatibility, and potential in cartilage regeneration. ¹⁴ In various evaluated studies, MSCs have been sourced from different origins as summarized in Table 1. The prevalence of animal-derived cell sources may be attributed to the study design involving combined in vitro and in vivo studies, in which cells from the same species are more easily integrated with scaffolds implanted in congruent animal models because of heightened immune compatibility. However, some studies reveal positive results regarding immune compatibility by using a different-source AM with cells from different species. ^39,41,88

Thus, combining different immune responses between different donors of AM could be important factors for further exploration of AM-based therapeutics. Animal models are considered a good model to evaluate comparable conditions in humans. ⁸⁹ Nevertheless, a lack of studies in large animals poses a challenge, as such models are crucial for evaluating implants intended for clinical application in the future. ^40,89

ADSCs are used in a fewer studies despite their similar capabilities and characteristics for cartilage tissue engineering as bone marrow-derived MSCs. ⁹⁰ The preference of MSCs may arise from the greater body of research evidence regarding tissue engineering coupled with MSCs, or the ease of accessing bone marrow in animal models. In human cell applications, adipose tissue offers a superior source of stem cells (ADSCs) because of its accessibility, quantity, and the relatively simple collection procedure compared with obtaining stem cells from human bone marrow or even the umbilical cord. ⁹¹

Limitations of this review process are the specificity of our search parameter focusing on AMs in cartilage regeneration. This narrow field may have resulted in a limited perspective of the entire field of research in cartilage tissue engineering. Also, by only relying on PubMed in our research process, some articles may have been oversighted. On the contrary, PubMed is one of the most significant sources of information in the field of medicine and its related sciences. In addition, PubMed includes references from other medical indices, allowing the identification of pivotal works or trends in cartilage engineering over the past years even through isolated usage. However, focusing on a specific aspect allows for a detailed overview and examination within a defined research topic. By including abstracts and their results in our review, efforts were made to minimize the bias of not capturing all the relevant articles available in full text.

CONCLUSION

Cartilage regeneration is a crucial part of research aimed at future clinical advancements. Recently, there has been a surge in attention toward ADM-based methodologies. Besides the development of synthetic scaffolds incorporating biopolymers and hydrogels, there is a rising exploration of ADM and ACM derived from various (animal) tissues. These matrices possess several advantages over purely synthetic scaffolds, including enhanced biocompatibility with reduced inflammatory reactions as well as possibly improved accessibility and reproducibility.

Our review emphasizes diverse approaches concerning AM-based therapeutics in research. There is considerable variation in scaffold materials and in vitro approaches, with a concurrent focus on incorporating various cell types. Recent trends in ACM-based engineering involve the development of biphasic scaffolds as well as the integration of fluid phases, such as hydrogels loaded with growth factors. These scaffolds are often constructed with AMs either synthesized or sourced from animal tissues, such as cartilage (ACM) or dermal (ADM) origins.

Analyzing study results reveals a notable success rate in both in vitro and in vivo settings, despite impending challenges. Key areas of focus include testing in large animal models, identifying suitable cell sources, ensuring optimal integration with the surrounding tissue by reducing inflammatory reactions and supporting chondrogenic growth support. Considerable obstacles remain before individually produced AM-scaffold compounds can be applied in broad clinical practice, also including high costs and limited availability of cell and scaffold sources. In addition, the heterogeneity and limited regenerative capacities of native cartilage tissue must be considered. Furthermore, mechanical and physical durability must be investigated before consideration of clinical application. Moving forward, research efforts should concentrate on developing easily reproducible scaffolds, ideally constructed from clinically validated and readily available commercial products. We suggest that in the future, the primary objective should not solely focus on repairing small cartilage defects but rather on achieving complete restoration of a fully defected joint or other cartilage sites by engineering osteochondral replacement. As we, in our research group, work on tissue engineering approaches as well, we would endeavor to use a combination of commercially (if possible) available AMs in combination with compound scaffolding, for example, 3D bioprinting for future research application. Furthermore, after collecting a wide range of information about several cells used in mentioned studies, we would propose to combine stem cells from easily accessible tissue such as fat tissue, with harvested chondrocytes to partly overcome the lack of cartilage tissue.

TAKE-HOME MESSAGES

Cartilage regeneration with AM-based therapeutic strategies could have a significant impact in clinical application.