Cartilage repair: unleashing PRP’s potential in organoid models

Abstract

Platelet-rich plasma (PRP) has emerged as a promising biological therapy in regenerative medicine due to its high concentration of growth factors and cytokines, which promote tissue healing and regeneration. In recent years, its application in cartilage tissue engineering has garnered significant attention. This study explores the synergistic interaction between PRP and cartilage organoids, a novel three-dimensional in vitro culture system that closely mimics the structural and functional properties of native cartilage. Cartilage organoids serve as a physiologically relevant model for studying cartilage development, disease progression, and regeneration. By integrating PRP with cartilage organoids, this review aims to enhance chondrogenesis, extracellular matrix synthesis, and cellular proliferation within the organoids. Emerging evidence suggests that PRP supplementation significantly improves chondrocyte viability, growth, and differentiation in cartilage organoids, thereby accelerating their maturation. This combination holds great potential for advancing cartilage repair strategies, providing a robust platform for preclinical studies, and paving the way for innovative therapeutic approaches for cartilage-related injuries and degenerative diseases. These key aspects—chondrogenesis, matrix synthesis, and cellular proliferation—were specifically selected due to their fundamental roles in cartilage tissue engineering and regeneration. Chondrogenesis is crucial for chondrocyte differentiation and maintenance, matrix synthesis ensures the structural integrity and functional properties of regenerated cartilage, and cellular proliferation supports tissue viability and repair. Addressing these factors is essential, as current cartilage regeneration strategies often suffer from limited long-term efficacy and inadequate extracellular matrix production. By elucidating the synergistic effects of PRP and cartilage organoids in these areas, this study seeks to bridge existing knowledge gaps and provide valuable insights for improving regenerative approaches in clinical applications, particularly for osteoarthritis and cartilage defects.

Introduction

Platelet-rich plasma (PRP) is a blood product containing a high concentration of platelets. The concept of “platelet-rich plasma” was first mentioned by Kingsley in 1954. In 1988, Knighton published the first series of PRP cases. Later, in 2001, Marks introduced a specific definition of PRP, stating that it contains approximately 1,000,000 platelets per microliter (Kingsley 1954; Mahoney et al. 1941; Marx 2001).

The use of platelet-rich plasma (PRP) is activated by calcium chloride or thrombin. The platelets in PRP release several growth factors, including platelet-derived growth factor (PDGF), transforming growth factor (TGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF) (Alsousou et al. 2009; King et al. 2002). Among the factors secreted by platelets in PRP, several play key roles in the growth and proliferation of cartilage. For example, TGF-β factors promote the formation of cartilage from stem cells, while PDGF helps maintain the phenotype of hyaline cartilage and also enhances proteoglycan synthesis (Joyce et al. 1990).

Normal cartilage tissue is typically 2-4 mm thick and consists of highly specialized cells called chondrocytes, embedded in a dense extracellular matrix (ECM). The reproduction of chondrocytes is limited, and their primary function is to produce and regenerate the ECM. Therefore, one approach to cartilage regeneration involves the use of stem cells and more effective stimulation of the main tissue cells. New tissue engineering solutions, such as microfluidics and 3D bioprinting, can be effective in regeneration and are currently more advanced than traditional three-dimensional organoid cultures for this purpose (Sophia Fox and Bedi 2009; Wei 2021).

Composition PRP

Platelet-rich plasma (PRP) is a concentration of platelets in plasma that has gained significant attention in regenerative medicine due to its potential to enhance healing processes. PRP is used across various medical fields, including orthopedics, sports medicine, and dermatology. Understanding the composition of PRP is crucial for optimizing its therapeutic efficacy (Pavlovic et al. 2016).

PRP is composed of several key components that contribute to its regenerative properties, including

Platelets are nucleated cells with a discoid shape, measuring approximately 2–3 µm in diameter. They play essential roles in regulating homeostasis, angiogenesis, and innate immunity. In an adult human, there are about one trillion platelets, which are renewed every 8–10 days. PRP typically contains 4–5 times the concentration of platelets found in whole blood. Additionally, platelets are small, anucleate cell fragments derived from megakaryocytes in the bone marrow (Thon and Italiano 2012).

Plasma is the liquid component of blood, consisting of water, electrolytes, proteins, and nutrients (Fig. 1). It serves as a medium for delivering platelets and their growth factors to the site of injury. Plasma is a complex mixture of water, mineral salts, and more than 1000 proteins and compounds, which can vary in composition based on the processes of synthesis, processing, and storage.Plasma contains essential proteins such as fibrinogen, which is critical for clot formation and wound healing, as well as albumin and globulins, which play important roles in immune responses and tissue repair (Fig. 1) (Liang et al. 2022).

Fig. 1.

Blood is composed of three main components: plasma (~ 55%), the buffy coat (~ 1%), and red blood cells (~ 45%). Plasma, the liquid portion of blood, is essential for maintaining homeostasis, transporting nutrients, and supporting immune function. It consists primarily of water (~ 90–92%), which acts as a solvent for biomolecules, facilitates the transport of cells, gases, and nutrients, regulates body temperature, and maintains blood volume and osmotic balance. Plasma proteins (~ 7–8%) include albumin, which maintains oncotic pressure and transports hormones, fatty acids, and drugs; globulins, which include immunoglobulins for immune defense and transport globulins for carrying ions, lipids, and fat-soluble vitamins; fibrinogen, which plays a crucial role in blood clotting by converting into fibrin during coagulation; and regulatory proteins, such as enzymes, hormones, and cytokines, that regulate physiological processes. Electrolytes such as sodium, potassium, calcium, chloride, and bicarbonate help regulate pH, osmotic balance, nerve signaling, and muscle function. Plasma also carries essential nutrients, including glucose, amino acids, lipids, and vitamins, which serve as metabolic fuel and building blocks, while waste products like urea, creatinine, and bilirubin are transported for excretion. Additionally, dissolved gases such as oxygen and carbon dioxide are present in plasma, supporting respiration and pH balance

The use of PRP therapy for treating cartilaginous lesions, including the different methods and optimal formulations of PRP, has yet to be fully established. However, the concentration of leukocytes in PRP is an important factor in the treatment of these lesions (Liang et al. 2022).

Because PRP contains all the components found in whole blood, the specific PRP protocol used can result in varying amounts of blood cells and leukocytes. In damaged tissue, neutrophils are the first leukocytes to migrate, playing a crucial role in tissue repair and the removal of microbes. The second category of leukocytes, monocytes, secretes MMP-2 (matrix metalloproteinase), MMP-9, and MMP-13, which are involved in the degradation of the extracellular matrix. This degradation facilitates cell migration and promotes better tissue healing (Wiesner and Vilcinskas 2010; Boswell et al. 2012).

Monocytes are also effective in secreting and providing growth factors, particularly those that promote cartilage development. Key factors include TGF-β1, PDGF, VEGF, IGF-1, and EGF, all of which play important roles in neoangiogenesis and tissue healing (Table 1). Neoangiogenesis refers to the formation of new blood vessels, which enhances cell nutrition, oxygen supply, and the formation of granulation tissue (Lingen 2001).

Table 1.

List of growth factors in PRP and their specific function (Pavlovic et al. 2016)

Growth factors	Functions
Transforming growth factors beta-1; TGFB1	Proliferation, and differentiation, in many cell types
Platelet-derived growth factor, alpha polypeptide; PDGFA	Potent mitogen for connective tissue cells
Platelet-derived growth factor, beta polypeptide; PDGFB	Promotes cellular proliferation, inhibits apoptosis
Platelet-derived growth factor C; PDGFC	Increase in the motility of different cells (mesenchymal cells, fibroblasts, smooth muscle cells, capillary endothelial cells, neurons)
Platelet-derived growth factor D; PDGFD	Plays a role in developmental and physiological processes and a disease like cancer, fibrotic diseases, and arteriosclerosis
Insulin-like growth factor I; IGF1	Mediates growth-promoting effects of growth hormone
Fibroblast growth factor I; FGF1	Induces liver gene expression, angiogenesis, and fibroblast proliferation
Epidermal growth factor; EGF	Induces differentiation of specific cells, acts as a potent mitogenic factor for a variety of cultured cells
Vascular endothelial growth factor A; VEGFA	Primarily a mitogen for vascular endothelial cells induces angiogenesis
Vascular endothelial growth factor B; VEGFB	Regulator of blood vessel physiology, a role in endothelial targeting of lipids to peripheral tissues
Vascular endothelial growth factor C; VEGFC	Angiogenesis and endothelial cell growth, and also affect the permeability of blood vessels

Function

Leukocytes play a crucial role in the immune response, helping to manage inflammation and infection. However, their presence can also result in increased inflammation, which may not be desirable in all therapeutic contexts (Zhang et al. 2024).

Anti-inflammatory properties of PRP

Inhibition of inflammatory cytokines

PRP plays a significant role in reducing joint inflammation by modulating the immune system and suppressing the production of certain pro-inflammatory cytokines. Studies have demonstrated that PRP decreases the expression of interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α), which are key cytokines involved in joint inflammation and tissue degradation. IL-1β and TNF-α contribute to the pathogenesis of degenerative diseases such as osteoarthritis by stimulating the production of matrix metalloproteinases (MMPs), leading to extracellular matrix breakdown and cartilage deterioration (Filardo et al. 2012). Additionally, PRP enhances the levels of interleukin-10 (IL-10), a well-known anti-inflammatory cytokine, which reduces synovial inflammation and inhibits intracellular inflammatory pathways. Increased IL-10 levels in synovial fluid following PRP injection may contribute to pain relief and improved joint function (Andia and Maffulli 2013).

Reduction of joint-degrading enzyme activity

One of the primary causes of joint tissue degradation in inflammatory joint diseases is the increased activity of matrix metalloproteinases (MMPs), which are responsible for extracellular matrix breakdown. These enzymes are upregulated by inflammatory cytokines such as IL-1β and TNF-α and contribute to cartilage erosion and diminished biomechanical properties of the joint. PRP mitigates this process by reducing MMP production. Research has shown that PRP decreases the levels of MMP-1 and MMP-3 in synovial fluid, thereby preventing the degradation of collagen and cartilage proteoglycans (Sundman et al. 2014). This protective effect not only slows disease progression but also creates a favorable environment for tissue regeneration.

Promotion of tissue repair and pain reduction

PRP contains various growth factors that promote tissue repair and regeneration. Key factors include transforming growth factor-beta (TGF-β) and platelet-derived growth factor (PDGF), which enhance collagen synthesis, support mesenchymal stem cell (MSC) differentiation, and improve the function of damaged tissue. Moreover, vascular endothelial growth factor (VEGF) in PRP promotes angiogenesis, enhancing blood supply to joint tissues, which accelerates the repair process (Gobbi et al. 2017). The combination of reduced inflammation and enhanced tissue regeneration contributes to pain relief in patients with osteoarthritis or tendon injuries. Clinical studies have indicated that PRP injections in patients with mild to moderate osteoarthritis significantly reduce pain scores (VAS) and improve joint function compared to conventional treatments such as hyaluronic acid (Raeissadat et al. 2020).

Inflammatory characteristics of PRP

Increased interleukin-6 (IL-6) levels

IL-6 is a multifunctional cytokine with both pro-inflammatory and anti-inflammatory effects. In some cases, PRP injection increases IL-6 production, potentially triggering acute inflammation. This increase is more pronounced in leukocyte-rich PRP (L-PRP) and may lead to synovial inflammation and pain exacerbation in certain patients (Dragoo et al. 2012). However, IL-6 can also contribute to tissue repair by promoting angiogenesis and tissue regeneration. Thus, its effects in PRP therapy depend on patient-specific factors, PRP composition, and disease stage.

Acute inflammatory response in the initial phase

A common reaction after PRP injection is a transient increase in pain and inflammation at the injection site due to immune system activation and the release of inflammatory mediators. This response is more evident in PRP preparations with high platelet and leukocyte concentrations, as these components can lead to the release of reactive oxygen species and inflammatory mediators (Sundman et al. 2014). In most cases, this reaction is temporary and subsides within a few days as PRP’s anti-inflammatory effects take over. However, in patients with advanced joint diseases, acute inflammation may lead to increased pain and restricted mobility.

PRP composition dependency

The inflammatory or anti-inflammatory properties of PRP are largely influenced by its composition. Studies have shown that leukocyte-rich PRP (L-PRP) induces a stronger inflammatory response compared to leukocyte-poor PRP (P-PRP). This difference is attributed to the presence of leukocytes in L-PRP, which increase the production of pro-inflammatory cytokines such as TNF-α and IL-6. In contrast, P-PRP has lower concentrations of these cytokines, resulting in more pronounced anti-inflammatory effects (Filardo et al. 2012). Therefore, selecting the appropriate PRP formulation based on the patient’s condition and joint pathology is crucial for optimizing therapeutic outcomes.

Method of preparation (extraction) platelet-rich plasma (PRP)

Platelet-rich plasma (PRP) is an innovative method that harnesses the body’s natural healing processes to treat injuries and accelerate wound healing. Various methods of PRP production yield different qualities of PRP. These differences affect not only the concentration of platelets but also the levels of red blood cells (RBCs), white blood cells (WBCs), cytokines, microvesicles, and other components (Arora et al. 2016; Amable et al. 2013).

The preparation of platelet-rich plasma (PRP) involves several precise steps that depend on the cellular contents, such as the levels of leukocytes and the amount of fibrin (Dhurat and Sukesh 2014). The following sections describe the commonly used methods:

The preparation method of PRP can affect the concentration of its components. Based on the cellular content and the structure of the extracted fibrin, PRP is divided into four categories:

1. Pure platelet-rich plasma (P-PRP): This category has a low leukocyte level.

2. Leukocyte-reduced platelet-rich plasma (L-PRP): This product has a high leukocyte content and low PRP, featuring a low-density fibrin network. Most commercial products fall into this category.

3. Platelet-rich fibrin (P-PRF): These products contain a high amount of fibrin and a low number of leukocytes. In these products, the fibrin fibers have a high density, and they are typically in gel form, making them unsuitable for injection.

4. A variation of L-PRP: This method produces results similar to those of the second group (L-PRP) but differs in having a higher density of fibrin (Dhurat and Sukesh 2014).

The method of separating blood components using density gradient centrifugation dates back to the 1960s and 1970s, when it was developed (Meer et al. 2022).

There are various methods for preparing PRP, but all of them begin with centrifugation. The two main preparation techniques are:

1. Open technique: In this method, blood is drawn from the patient’s body and processed under non-sterile conditions. Although all devices used in this technique are individually sterile, the overall process is not conducted in a fully sterile environment.

2. Closed technique: This method utilizes commercial devices and kits that are completely sterile, allowing for the preparation of PRP without the involvement of additional equipment (Alves and Grimalt 2018).

Single spin method

The single spin method is the simplest and most time-efficient technique for preparing PRP. It involves the following steps:

First step: blood collection

Depending on the needs and working conditions, 10–65 ml of blood is drawn from the patient.

Second step: centrifugation

In this stage, the collected blood is placed in a centrifuge, where it is spun at high speeds between 1,000 and 3,000 RPM for 5–15 min. This process separates the blood components based on their density and weight.

Third step: plasma extraction

After centrifugation, the blood will have three layers: red blood cells at the bottom, platelet-poor plasma (PPP) at the top, and a buffy coat in the middle containing platelets and white blood cells. The top layer of PPP and the buffy coat are carefully extracted (Muthu et al. 2022).

Single spinning changes 1–3 times the amount of platelet concentration compared to the initial amount of blood (Mikel et al. 2012).

Double spin method

The double spin method is more complicated and time-consuming, but it yields a higher concentration of platelets. Generally, a centrifuge is used to prepare PRP; however, different techniques and protocols can influence the heterogeneous nature of PRP and its commercial formulations.In the first phase of centrifugation, a lower relative centrifugal force (G-force) is applied, which allows the red blood cells to separate while keeping the platelets suspended. In the second phase, a higher G-force is applied for a longer duration, which determines whether the platelets are removed or retained in the final product (Jo et al. 2013). The steps of which are as follows:

First centrifugation

Blood collection

Blood is collected from patients according to the required amount.

Initial centrifugation

In this method, the blood is centrifuged at a lower speed (approximately 1200 RPM) for 10–15 min to form three main layers.

Plasma separation

Similar to the single spin method, the plasma and buffy coat are collected, while the remaining blood is drained.

Second centrifugation

The plasma and buffy coat collected in the first stage are placed in the centrifuge for a second round at about 3,000 RPM for another 10–15 min. This step allows for the removal of more platelets.

Final PRP extraction

The plasma containing the concentrated platelets is extracted, leaving behind the final PRP product (Muthu et al. 2022).

Double spinning results in a platelet concentration increase of 4–8 times compared to the original blood volume. Additionally, this method significantly reduces the presence of leukocytes, and the platelet concentrates obtained are referred to as high platelet-rich plasma (P-PRP) (Mikel et al. 2012).

Gel separator method

About 30 years ago, Becton, Dickinson, and others first utilized these gels. These gels are special polymers with a specific density that create a layer between serum and plasma (Bowen et al. 2010; Schouwers et al. 2012). Gel separator tubes facilitate the rapid separation of serum and plasma from cellular components and offer both advantages and disadvantages. Among the advantages are their effectiveness in preventing cell hemolysis and increasing the stability of analytical results (Table 2), which benefits laboratory personnel, as they are also easy to handle. However, a significant disadvantage is that gel separator tubes can affect the concentration of drugs and cellular components. When cells and compounds are exposed to the gel for an extended period, the concentration of drugs can significantly decrease in laboratory conditions, which is not desirable (Karppi et al. 2000; Dasgupta et al. 1994). Additionally, gel compounds can be effective in separation; for example, the lack of interaction between lipophilic compounds and the gel aids in the separation process (Schouwers et al. 2012; Bowen 2014). For this reason, suppliers of laboratory diagnostic reagents (IVD) avoid using gel separator tubes for medicinal purposes (Hepburn et al. 2016; Borai et al. 2016).

Table 2.

Advantages and disadvantages of gel separator method

Advantages	Disadvantages
Quick and complete separation of serum/plasma from blood cells	Adsorption of analytes on the polymer gel
Increase in serum yield	Elusion of materials
Possibility of direct use of the primary tube on an analyser	Promotes cellular proliferation, inhibits apoptosis
Diminishing sample manipulations	May alter blood composition
Liminating potential contamination with hazardous substances	Hence affecting laboratory results
Decreasing hands-on time	Its therapeutic use is controversial and unreliable
Reducing the chance of sample switching
Karppi et al. (2000); Bush et al. (2001); Dasgupta and Yared (2000); Bergqvist and Eckerbom (1984); Datta PJCb. (1998); Dasgupta and Blackwell (1996)	Quattrocchi et al. (1983); Hilborn (1987); Toffaletti et al. (1984); Hammett-Stabler (2024)

The gel separation method is a specialized technique for separating PRP from blood cells during centrifugation. The steps include:

Blood collection

The collected blood is placed in tubes containing separating gel.

Centrifugation

Based on pre-determined protocols, the centrifugation speed and time are set. During centrifugation, a barrier is formed between the red blood cells and plasma.

Plasma extraction

This barrier allows the PRP to be extracted from the top of the gel, minimizing contamination from red blood cells and plasma.

Among the advantages of this method are reduced contamination from red cells and its simplicity. However, the level of platelet concentration depends on the specific gel used (Steuer et al. 2016).

Apheresis method

This method is suitable for collecting blood products such as red and white blood cells, platelets, and plasma. In this process, platelets are separated from whole blood, and after collecting the donor’s blood and isolating the platelets and plasma, the remaining blood components are returned to the donor’s body (Javadzadeh Shahshahani et al. 2023).

Platelets collected from a donor are referred to as monolithic platelets. They are gathered through a process called plateletpheresis, which typically takes 1–2 h. The collected platelets usually contain 5–6 times the normal concentration of platelets found in whole blood (Meer 2012).

The apheresis method, commonly used in clinical and laboratory settings, involves the use of an apheresis machine that continuously collects and processes blood) (Meer 2013). The procedure includes:

Blood collection and processing

Blood is drawn from the patient and passed through an apheresis machine.

Component separation

The machine separates the blood components in real-time, isolating the platelets and plasma from red blood cells and other elements.

Collection

The PRP is collected directly from the machine, resulting in a highly concentrated product.

The apheresis method allows for precise control over the platelet concentration and the volume of PRP collected, making it ideal for specific medical and research applications (Zingsem et al. 1998; Javadzadeh Shahshahani and Akhavan Tafti 2023).

Organoid models

Despite progress in cartilage tissue engineering, the diverse methods for obtaining cartilage organoids remain underexplored. Techniques such as hanging drop culture, microstructured systems, and dynamic bioreactors offer distinct advantages but have not been comprehensively compared or integrated. Mechanical and electrical stimulation further enhance chondrogenesis, yet their role in organoid formation is not well understood.

Dynamic bioreactors

Dynamic bioreactors are designed to create physiological conditions similar to the in vivo environment during chondrogenesis. These systems can regulate nutrient flow, mechanical stimulation, and other environmental factors to enhance the growth and differentiation of chondrocytes. Dynamic bioreactors include the following types:

Rotating bioreactors

These bioreactors use rotational motion to uniformly mix cells and distribute nutrients. This method reduces localized shear stress and promotes chondrocyte differentiation. Rotating bioreactors are commonly used to create dense and uniform three-dimensional structures (Wendt et al. 2005; Haasper et al. 2008).

Compression bioreactors

In these systems, periodic compressive forces are applied to scaffolds containing chondrocytes. These forces mimic the natural pressures found in joints, leading to increased production of glycosaminoglycans and type II collagen in the extracellular matrix (Wendt et al. 2005).

Tensile bioreactors

This type of bioreactor applies cyclic tensile forces to biological scaffolds. Tensile stimulation enhances the expression of genes associated with chondrocyte differentiation and strengthens the mechanical properties of the engineered tissue (Martin et al. 2004; Xu et al. 2015).

Mechanical and electrical stimulation

Physical methods, such as mechanical and electrical stimulation, are widely used to improve chondrocyte differentiation and enhance the biomechanical properties of cartilage tissue. These methods include the following subcategories:

Mechanical stimulation

This approach applies external forces such as tension, compression, or shear stress to cells and scaffolds.

• Compressive loading: Applying cyclic compression to scaffolds enhances chondrocyte gene expression and increases extracellular matrix production (Wren and Beaupre 2000; Main et al. 2020).

• Tensile loading: This method applies controlled mechanical tension to cells, improving cartilage tissue density and mechanical strength (Gilbert and Blain 2018).

• Shear stress: Fluid flow-induced shear stress enhances chondrocyte viability and improves nutrient distribution (Chowdhury et al. 2021; Verbruggen and Vaughan 2012).

Electrical stimulation

Electrical stimulation involves applying external electric fields to regulate the expression of chondrogenesis-related genes. This method includes:

• Direct current stimulation: Weak electric fields enhance ion channel activity and induce signaling pathways associated with chondrogenesis.

• Pulsed electrical stimulation: Intermittent electrical pulses promote the proliferation and differentiation of mesenchymal stem cells into chondrocytes.

• Electromagnetic field stimulation: Controlled electromagnetic waves stimulate proteoglycan and collagen production while preventing cartilage degradation (Vaiciuleviciute et al. 2023; Zimmermann et al. 2024).

Hanging drop culture

Hanging drop culture is a simple yet effective method for generating cartilage organoids without using scaffolds. In this technique, cells are suspended in a droplet and self-organize into a three-dimensional structure under gravitational forces (Chen et al. 2020). This technique includes the following aspects:

Standard hanging drop culture

The hanging drop culture technique is a widely used method for generating three-dimensional (3D) cellular aggregates, such as spheroids and organoids, without the need for a solid scaffold. This method relies on the natural tendency of cells to aggregate under the influence of gravity and cell–cell adhesion forces, forming compact structures that mimic native tissue architecture. To perform this technique, a small volume of cell suspension (typically 10–30 µL) containing a defined number of cells is placed as individual drops on the underside of a culture dish lid. When the lid is inverted, the drops remain suspended due to surface tension, and the cells within each drop settle and aggregate at the lowest point, forming a spherical cluster over time. This environment prevents cell attachment to any surface, promoting self-organization and cellular differentiation. Hanging drop culture is commonly used in tissue engineering, regenerative medicine, drug screening, and developmental biology. Additionally, it provides a controlled microenvironment for studying cell interactions, differentiation, and organoid formation, including cartilage tissue engineering, where mesenchymal stem cells (MSCs) or chondrocytes can self-assemble into cartilage-like structures. However, limitations such as nutrient diffusion constraints and hypoxia in larger spheroids necessitate further optimization or integration with bioreactors for improved viability and functionality (Martinez et al. 2008).

Microstructured culture systems

Microstructured culture systems utilize microwell plates and microfluidic devices to guide the formation of cartilage spheroids with uniform size and structure. These methods include:

Microwell plates

This approach uses small wells to direct cell aggregation and facilitate the formation of three-dimensional organoids. Each well acts as an isolated chamber, ensuring uniformity in size and cellular composition (Futrega et al. 2015).

Microfluidic systems

Microfluidic channels precisely control the culture environment and nutrient distribution for cells. These systems are classified as:

Static microfluidic systems

In these systems, cells are confined within microfluidic channels, where they grow and differentiate (Tolabi et al. 2023).

Dynamic microfluidic systems

Continuous media flow enhances gas exchange and removes metabolic waste from the cell culture environment (Liu et al. 2022).

Development of organoid models

Various methods have been developed for creating models to study human organs, including the use of stem cells in two-dimensional cultures with or without extracellular matrix (ECM), three-dimensional biopatch techniques, and microfluidic devices. These methods were established prior to the development of organoid models, which have been utilized in pharmaceutical studies and pathological research. However, organoids represent a unique and advanced approach to study human tissues because they possess the ability to self-organize in a manner similar to the original tissue. In many cases, the engineered tissue is indistinguishable from the native tissue (Sato et al. 2009, 2011; Dortaj et al. 2024).

Animal models are the most common methods for experimental applications; however, they do not offer a comprehensive understanding of biology and diseases. Organoids, on the other hand, are three-dimensional culture systems composed of stem cells or mature tissue cells. They have the ability to replicate the architecture of the original tissue while imitating its intricate details. Organoids provide valuable opportunities to study human diseases in conjunction with animal models. They can be utilized to investigate a variety of conditions, including orthopedic diseases, infectious diseases, and genetic disorders (Kim et al. 2020).

Applications of organoid models

Organoids are practical options for modeling and examining tissue growth, as they provide conditions conducive to homeostasis and regeneration. They are particularly valuable in genetic studies and investigations of mutations, allowing for research that cannot be conducted on a fetus in the body (Fig. 2) (Hofer and Lutolf 2021).

Fig. 2.

Organoids are 3D miniaturized versions of human tissues and organs that closely mimic the in vivo environment, making them a valuable tool in various research fields. They offer an advanced alternative to traditional animal models, addressing the physiological differences between species. In disease modeling, organoids can be derived from patient-specific induced pluripotent stem cells (iPSCs) to study genetic diseases, cancers, and infectious conditions like cystic fibrosis. For example, cystic fibrosis organoids allow researchers to investigate mutations, gene therapy, and drug responses. In cancer research, tumor organoids help explore tumor growth, invasion, and treatment responses, enabling more personalized therapies. Organoids also facilitate drug screening by offering a high-throughput platform for testing the efficacy and toxicity of new and existing drugs, including those withdrawn from the market. Additionally, they are key in personalized medicine, providing insights into individual drug responses based on genetic diversity. Organoids contribute to developmental biology by modeling organogenesis and investigating the effects of genetic and environmental factors on human development. Lastly, they hold promise in regenerative medicine, offering a potential source of cells for tissue engineering and therapeutic applications, such as retinal transplants

Disease modeling

Organoids can simulate conditions similar to those of body tissues and organs, making them a more suitable alternative to animal models in laboratory settings. They provide a model that closely resembles the human body. Even when suitable animal models are available, physiological differences between animals and humans can affect experimental outcomes. The most significant contribution of organoids is their ability to bridge the gap between human and animal models. Useful applications of organoids can be found in conditions such as cystic fibrosis and polycystic kidney disease (Dekkers et al. 2013; Xia et al. 2013). Organoids provide a valuable platform for studying the pathogenesis of various diseases. Researchers can generate organoids from patient-derived induced pluripotent stem cells (iPSCs) to model genetic diseases, cancer, and infectious diseases. For example:

Cystic fibrosis

Intestinal organoids derived from cystic fibrosis patients can be utilized to study the impact of mutations on epithelial function (Conti et al. 2022). An organoid model of cystic fibrosis can be created from cells of the colon, liver, respiratory system, small intestine, or adult stem cells derived from humans or mice. Additionally, cystic fibrosis organoids can be utilized to investigate gene therapy interventions and examine the effects of specific mutations. The cystic fibrosis transmembrane conductance regulator (CFTR) is present in organoids, such as those derived from the intestine (Goor et al. 2011; Berkers et al. 2019).

Cancer

Tumor organoids can be created to study tumor growth, invasion, and response to therapies. Another organoid model for cancer can be derived from biopsies of tumors located in the digestive tract, liver, lung, and breast. The use of organoids demonstrates their ability to represent a variety of diseases, making them excellent and advanced tools for personalized medicine at the bedside. Additionally, organoids provide high-throughput screening capabilities and accurate toxicity assessments (Fan and Demirci 2019; Clevers 2019).

Drug screening and development

The use of organoids as a system for drug screening allows researchers to determine the most beneficial drugs for patients, facilitating drug discovery and development. In contrast to traditional models such as animal studies and clinical trials, which require high costs and lengthy approval processes, organoids can provide a more efficient alternative. They can also be used to test and validate drugs that have been withdrawn from the market for various reasons, including adverse reactions in some patients. However, organoid systems for drug screening need to be optimized to ensure feasibility and must be capable of creating a suitable environment that closely resembles the human body (Kondo and Inoue 2019; Miranda et al. 2018).

Personalized medicine or precision medicine

Genetic diversity among humans significantly influences the severity and prevalence of diseases, as well as variations in drug responses. One of the primary functions of organoids is to utilize a patient’s own genetic material to create precise and tailored treatments, a concept known as precision medicine or personalized medicine. Organoids enable the selection of the most appropriate drug and dosage for individual patients. This technology facilitates the identification of effective drugs across a wide spectrum of phenotypes, making it particularly effective for screening drugs aimed at targeting specific patient profiles (Rossi et al. 2018).

Developmental biology

Organoids offer valuable insights into human development by mimicking the processes of organogenesis. Researchers can study early developmental stages and examine the impact of genetic and environmental factors on development. While many cells in the organoid system exhibit functional capabilities, pluripotent stem cells (PSCs) serve as particularly valuable models in developmental biology studies (Kanton et al. 2019).

Regenerative medicine

Several reports have indicated that organoids can serve as a foundation for advanced therapies, as they provide suitable cell sources for tissue engineering and cell therapy in human applications. For example, the use of embryonic stem cells (ESCs) in retinal transplantation highlights the significance of cells that can be derived from organoids (Shirai et al. 2016).

Significance and future directions

Advancements in research

Organoids hold great promise for advancing cell proliferation and tumor models that elucidate biological mechanisms, pathogenesis, and disease treatment. These models provide a more accurate representation of in vivo tissues compared to traditional two-dimensional cell cultures and have the ability to encapsulate human-specific biology in a dish. The lack of reproducibility in micro-based technologies presents promising solutions to address many current challenges in organoid production. Techniques such as micropatterning and microfluidic platforms allow for precise control over cell structure size and shape.Microtechnology solutions can enhance the reproducibility of organoids and facilitate nutrient delivery and exchange, the induction of mechanical cues like shear stress, and real-time monitoring of growth and environmental parameters through sensors. Moreover, microtechnology techniques are conducive to mass production, which is essential for drug testing and commercial applications. The future of organoids, aided by microtechnology, will depend on the integration of multiple organoids into a single platform to replicate the complex cellular microenvironment observed in vivo. Another exciting area for future exploration is the assembly of organoids from different tissues to form functional units of the human body, known as assembloids, which will allow us to study the interactions of human tissues in vitro (Velasco et al. 2020a).

Ethical considerations

Organoids reduce the reliance on animal models, addressing ethical concerns related to animal testing. They offer a humane and potentially more accurate alternative for studying human diseases and drug responses (Mollaki 2021).

Source of stem cells

In organoid and organ-on-a-chip models, various cell sources from both human and animal origins are used. These sources can be categorized into two groups: pre-existing differentiated cells (such as primary cells, immortalized cell lines, and tissue biopsies) and undifferentiated cells (such as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and adult stem cells (ASCs)), each with distinct characteristics. Animal cell sources differ from human cells in their physiological complexity and are unable to replicate the genetic mutations and variations inherent in human cells for disease modeling and drug screening. Pre-existing differentiated cells are well-established sources that are widely available, exhibit genetic homogeneity, and show minimal phenotypic discrepancies compared to actual tissues. On the other hand, undifferentiated cells can successfully differentiate into specific lineages, but they face challenges such as arrested differentiation, unclear disease phenotypes, and potential mutagenesis or carcinogenicity due to epigenetic alterations or viral-induced DNA methylation changes. Therefore, selecting the appropriate cell source based on the research goals and genetic background is crucial in both organoid and organ-on-a-chip systems (Hadley et al. 2020; Yesil-Celiktas et al. 2018).

Different cell sources, such as embryonic tissues, adult tissues, ESCs, and iPSCs, play crucial roles in the production of organoids. ESCs are primarily obtained from various sources, with the most well-known method involving the extraction of the inner cell mass from fertilized blastocysts under laboratory conditions. However, a significant concern surrounding these cells is the ethical implications related to fetal life. Different cultural and religious perspectives attribute various moral values to the status of the fetus, leading to substantial ethical debates regarding the use of ESCs in organoid technologies. The developmental stage of the embryos or ESCs used in organoids raises moral considerations, yet the use of these cells is governed by strict protocols, including informed consent and appropriate measures to ensure ethical compliance. Beyond the ethical concerns regarding cell use, factors such as potential health risks for donors and agreements on compensation for egg donation also contribute to the complexities of these ethical issues. In contrast, iPSCs serve as suitable alternatives to ESCs in organoid research, as they help mitigate many of the ethical concerns associated with the use of embryonic stem cells (Jones 2016).

For example, a liver-kidney-on-a-chip seeded with Hep RG cells significantly differs from one seeded with HepaG2/C3A cells, as only the former can metabolize ifosfamide into the toxic metabolite chloroacetaldehyde. Additionally, ESCs or iPSCs-derived organoids differ noticeably from primary tissue-based organoids, particularly in terms of the presence of mesenchymal cell types and the cultivation process. Therefore, selecting the appropriate cell source in line with the research objectives and genetic context is crucial for ensuring accurate and reliable outcomes in organoid and organ-on-a-chip technologies (Eiraku et al. 2011).

Organoid models are generally derived from primary tissue biopsies or stem cells to model the structure and functionality of organ tissues. The most common stem cell types, including ASCs, ESCs, and iPSCs, have the essential ability to differentiate into specialized cells or clusters of cells and assemble into intricate structures resembling their counterparts in vivo. ASCs can be derived from various sites of the human body in both juveniles and adults, making them easy to access. ASC-derived organoids typically display a more mature phenotype and have been employed to model various organs (Hadley et al. 2020; Jones 2016). However, ASCs can only differentiate into certain types of cell lineages depending on the derived tissues and have various protocols that result in heterogeneous phenotypes of organoids from batch to batch, making them less applicable as a cell source for organoids compared to other stem cells (Yesil-Celiktas et al. 2018).

ESCs originate from the inner cell mass (ICM) of an early-stage embryo called the blastocyst. Both ESCs and ICM cells are pluripotent or totipotent cells. Notably, one of the earliest types of complete organoids originates from mESCs (mouse embryonic stem cells) by culturing them under floating conditions within mixed culture media. The most significant advantage of ESCs is their unlimited differentiation potential and high phenotype fidelity. However, research associated with ESCs raises ethical difficulties and requires rigorous regulation, making it difficult to apply this cell source in widespread practical research today (Eiraku et al. 2011). In addition to ethical concerns, there are still technical obstacles in guaranteeing the creation of various cell lines with genetic diversity, as well as developing well-defined protocols for directional differentiation, which further hinder the application of ESCs (Thomson et al. 1998).

iPSCs represent significant advancements in transfection protocols for stem cells and provide an ideal cell source for organoid models. iPSCs can be conveniently generated from cells harvested from specific tissues under defined factors, by inducing dedifferentiation into the particular lineage and genome. Furthermore, iPSCs can be easily harvested from donors with specific diseases, without ethical concerns, making them highly applicable to studies on organ-level dysfunctions (Scott et al. 2013). The most advanced techniques (e.g., CRISPR/Cas9 genome editing technology) can be employed to provide unique opportunities for delving into human diseases. However, some hurdles still exist in the application of iPSCs, such as the arrest of their differentiation and variations in disease phenotypes that may result from differences between the tissue of origin and genotype. Therefore, future efforts will focus on developing more reliable protocols for stem cell differentiation and eliminating mutations, chromosomal abnormalities, and epigenetic variations in the DNA of stem cell sources (Snyder et al. 2011).

Informed consent of cell donors

Induced pluripotent stem cells (iPSCs) offer a promising alternative that helps mitigate the ethical concerns associated with the use of embryonic stem cells (ESCs). However, iPSCs also present complex ethical challenges, particularly regarding the informed consent of tissue donors. From a legal perspective, the informed consent process must prioritize the protection of donors’ rights, ensuring their autonomy and self-determination are respected. This involves obtaining explicit agreement from donors not only for the use of their cells in research but also for the potential development of standard and advanced treatments that may arise from such research (Mollaki 2021).

Issues specific to brain organoids

Investigating brain diseases poses significant challenges, primarily due to the physiological and functional differences between the human brain and those of other species. Ethical considerations further complicate studies involving the human brain, particularly in fetuses, making such research difficult. Brain organoids present a valuable tool for exploring brain-related conditions. However, a major concern surrounding brain organoids is their potential to acquire human cognitive features, including the ability to recognize sensations and respond to stimuli. Additionally, another challenge is whether the use of original brain cells retains the donor’s memories or if these memories are entirely absent in the resulting organoid (Lancaster et al. 2013; Lavazza 2018, 2020).

Issues specific to gonadal organoids

Similar to other organoid models, testis and ovary organoids derived from iPSCs hold promise in reproductive system diagnostics, including toxicology, pathology, and biology. These organoids can serve as a valuable cell source in cases of infertility, and male gamete donation can mitigate ethical concerns related to genetic and hereditary transmission due to genetic differences from the parents. However, as with other methods, the use of reproductive organoids necessitates the informed consent of donors to address potential ethical issues (Heidari-Khoei et al. 2020).

Issues specific to multi-organoid complexes

The community of multiple organoids or their complexes presents a promising avenue for drug testing, drug discovery, and personalized medicine. However, this approach raises significant ethical concerns. The biological functions of these organoid complexes can evoke ethical reactions regarding their ability to exhibit suitable functions or behaviors. Given their capacity to respond to stimuli and display independent behavior, organoid complexes can attract moral scrutiny (Achberger et al. 2019; Xiang et al. 2019).

Gene editing

In the study of human diseases, the integration of organoid systems with gene editing techniques can enhance diagnosis and treatment. Gene editing allows for the acquisition of rare genotypes that can be utilized in organoids. Individuals with unique genotypes may provide valuable contributions to organoid research, but they often face significant moral pressure when it comes to donating their cells. Utilizing gene editing on organoids derived from special genotype cells can help mitigate these ethical concerns. However, the most pressing ethical issues related to gene editing in organoids arise from the potential transplantation of altered cells into the human body. This includes concerns regarding the modification of germ cells and embryos, as well as the implications of intervening at early stages of human development, which can challenge the notion of human dignity (Lavazza 2019; Hendriks and Clevers 2020).

Organoid transplantation

Organoid technology has been proposed as a potential source for organ transplants, although many experts believe that achieving this goal is still a distant prospect. Transitioning from laboratory research to clinical applications, human organoids could help address the global shortage of grafts needed for replacing damaged tissues or entire organs. However, before organoids can be utilized clinically, they must meet specific criteria concerning size, maturity, functionality, and safety. Continuous research and advancements in organoid technology are expected to gradually overcome these challenges. Recently, Liu et al. demonstrated the ability to upscale mini-organs using Multi-Organoid Patterning and Fusion, successfully creating scaffold-free airway tubes with defined shapes. This indicates that size may not be a limitation for future organ transplant applications (Liu et al. 2021).

Before progressing to clinical trials, it is crucial to address the ethical considerations associated with a first-in-human organoid transplantation trial. These issues are common in all initial human trials involving new therapeutic methods. A first-in-human trial for organoid transplantation would require extensive preclinical research, including studies using human-animal chimeras, to provide sufficient evidence of organ integration, functionality, and safety. The trial must thoroughly evaluate potential benefits and risks, ensure a favorable risk–benefit ratio, and involve careful selection of participants, accompanied by robust informed consent procedures. A unique aspect of these trials is the involvement of vulnerable patients in Phase I, who are often in the late stages of disease and urgently require transplants. This scenario raises the risk of “therapeutic misconception,” where participants may mistakenly believe they will receive direct therapeutic benefits from the trial, rather than simply contributing to scientific knowledge. Therefore, it is essential to ensure that participants fully understand the nature and potential benefits of the research during the informed consent process (Henderson et al. 2007).

Commercialization of organoids

In terms of commercial importance, the presence of tissues and cells surpasses that of transplantation and blood transfusion, highlighting the need for ethical clarity regarding their use in organoid systems, disease modeling, and regenerative medicine. The commercialization of human tissues and organs presents significant ethical and legal challenges, particularly concerning property rights. It is essential to establish clear regulations and laws regarding the ownership of tissues created from the human body, as well as to address the financial interests involved. These measures are necessary to resolve the ethical dilemmas associated with the commercialization of human parts and tissues (Boers and Delden 2019; Lenk 2012).

Patentability of organoids

The possibility of registering organoid systems is a significant aspect of their commercialization. Patenting serves to protect intellectual property and encourage scientific innovation. Organoids, which are derived from human cells, are often considered patentable inventions. In some countries, such as Japan, rights have been granted to those who register organoids as patents. However, whether an organoid qualifies for patent protection depends on its specific characteristics and whether it meets the necessary criteria (Smadar and Dvir-Ginzberg 2016).

The cost of treatments and issues of equity

The high cost of treatment methods, such as cell therapy, can be attributed to the commercialization and patentability of organoids. This situation raises serious ethical concerns about the potential for inequality in access to these treatments. It is crucial to clearly define the general accessibility of organoid systems for everyone, as well as to ensure equitable sharing of benefits among the stakeholders involved in this technology (Gonçalves 2020; Huang et al. 2019).

Misuse and dual use issues of organoids

The potential for dual use and malicious applications of organoid models must be considered, as is the case with all technologies. As organoid systems continue to advance, the establishment of strict regulations to prevent misuse becomes increasingly important. While researchers generally have humanitarian intentions in their use of technology, this alone does not guarantee ethical practices, as these facilities could also be exploited by malicious individuals. Currently, there is a prevailing opinion that specific regulations addressing dual and malicious uses of organoids are unnecessary, as existing laws already provide a framework for oversight (Rinaldi and Colotti 2019).

Organoid biobanking

Organoid biobanks are essential for research, as they create repositories with living structured systems that store tissues, cells, and even organs, contributing significantly to scientific research and development. However, organoid biobanks face numerous ethical issues due to the nature of organoids. Some of these concerns have been previously identified, while others are emerging. One of the most pressing problems with organoid biobanks today is the absence of laws and principles or mandatory regulations that clearly define the rights of donors and consumers (Lensink et al. 2021).

Challenges associated with the integration of PRP into cartilage organoid models

While organoid technology has advanced rapidly, several challenges still remain:

Complexity and maturation

None of the current organoid systems can fully reproduce the physiological conditions of all types of body cells, nor can they replicate the maturation and function of entire organs. However, these organoids do produce essential tissue-specific functions, which are highly relevant to this system. Additionally, most organoid models do not include all types of tissue cells; they typically involve only tissue-specific cells, such as specialized mesenchymal, immune, or nerve cells, in the creation of the organoid model (Cordero-Espinoza et al. 2021; Takebe and Wells 2019).

Reproducibility

One of the most significant challenges facing organoids is the inability to control their size, which hinders the reproducibility of models. Without a standard and reproducible model, examining the microenvironment and extracellular matrix (ECM) becomes difficult (Kelava 2016). In multicellular and multitissue systems, reproducibility is challenging due to the difficulty in coordinating proliferation and differentiation among various cell types (Andersen et al. 2020; Marsee et al. 2021). Controlling the heterogeneity of organoid systems in high-throughput screening applications is crucial, as it affects reproducibility; moreover, achieving imaging with adequate spatial and temporal resolution becomes challenging (Toh et al. 2015, 2011).

Scale-up and accessibility

As is well known, more complex systems tend to have larger structures, while simpler models with smaller dimensions provide a more accurate representation of the overall structure and performance (Andersen et al. 2020). Access to organoids is often limited due to their stability and maintenance duration. This inaccessibility reduces the ability to control different parts of the organoid. For instance, in epithelial organoids, such as those derived from the intestine, access is required on both the luminal (inside) and basolateral (outside) sides of the epithelium. To illustrate this, consider the interaction between pathogens and cells: microbes can be delivered to the apical side of the organoid lumen, while metabolites and cytokines are delivered to the basolateral side (Lancaster et al. 2013; Qian et al. 2016). One of the challenges of organoids is their size and geometrical shape, as well as the difficulty in controlling the size and number of their cells. Due to the high diversity among organoid models, mass production becomes problematic, which can adversely affect their applications, such as drug screening (Lancaster et al. 2013).

Morphological and functional

Various factors influence the maturation and function of organoids. For instance, the supply of nutrients and the presence of dead cells within the lumens of organoids can significantly affect their development. These two factors are particularly impactful on induced pluripotent stem cells (iPSCs) and their functionality. As organoids grow larger, it becomes increasingly difficult for the cells in the center to receive adequate nutrients, which can lead to cell death. This phenomenon is especially common in organoids with a more compact structure, such as brain organoids. In contrast, pancreatic organoids, which possess a lumen, can accumulate dead cells within that lumen, leading to the degradation of the organoid. One potential solution to this issue is the mechanical fragmentation of the organoid (Xiang et al. 2000). Organoids derived from pluripotent cells effectively generate various types of cells and perform essential cellular and organ functions. However, they exhibit limitations in replicating the morphology and functionality of adult tissues (Hofer and Lutolf 2021).

Applications of PRP in cartilage organoid models

Platelet-rich plasma (PRP) is gaining considerable attention in regenerative medicine, particularly regarding cartilage repair and the development of cartilage organoid models. Derived from a patient’s own blood, PRP contains a high concentration of growth factors and cytokines that are critical for tissue regeneration and healing. Its applications in cartilage organoid models are multifaceted and promising (Bai et al. 2023), as outlined below:

Enhancing chondrogenesis

PRP is rich in growth factors such as platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-β), and insulin-like growth factor (IGF). These factors play a crucial role in enhancing chondrogenesis, the process by which cartilage is formed. When applied to cartilage organoid models, PRP can significantly boost the differentiation of stem cells into chondrocytes, the cells responsible for cartilage formation. This enhancement is vital for creating more robust and functionally relevant cartilage tissues in vitro (Table 3) (Kabiri et al. 2014).

Table 3.

Different cell type and model culture for PRP extraction

Cell type	Classification of PRP	PRP application to culture	Model cultures	Outcome	References
Porcine chondrocytes	P-PRP	10% PRP	3D cultures	Improve cell proliferation and matrix biosynthesis, PG and collagen synthesis	Akeda et al. (2006)
New Zealand white rabbit chondrocytes	P-PRP	20%	3D cultures	Improve cell proliferation and increase aggrecan, BMP-2, BMP7, col-II expression in the long-term	Park et al. (2012)
Rabbit mesenchymal stem cells (MSCs) from bone marrow (BMSC) and from adipose (ADSC)	PRP	16%	3D scaffold	Higher cartilage-specific gene and protein expression as well as subchondral bone regeneration	Xie et al. (2012)
Japanese white rabbit chondrocytes	P-PRP	3%	PRP with alginate beads	Increase chondrocyte GAG synthesis and maintain cell phenotype	Saito et al. (2009)
New Zealand White rabbit	P-PRP	0.1–20%	Chondrocyte cultures	Chondrocyte proliferation and differentiation	Park et al. (2012)

P-PRP pure platelet-rich plasma, PG proteoglycans, GAG glycosaminoglycans, 3D three dimensional, BMP bone morphogenetic protein, collcollagen

Improving organoid maturation

Cartilage organoid models aim to replicate the complex structure and function of natural cartilage. PRP contributes to the maturation of these organoids by providing a supportive environment that promotes cellular proliferation and extracellular matrix (ECM) production. The growth factors in PRP stimulate the synthesis of collagen and proteoglycans, which are essential components of the cartilage ECM, thereby enhancing the structural integrity and biomechanical properties of the organoids (Moroz et al. 2013). Platelet-rich plasma (PRP) can be utilized to stimulate the treatment of cartilage injuries or to provide an environment conducive to the growth of cartilage organoids. PRP activates specific signaling pathways essential for tissue regeneration and the formation of organoids that resemble mature tissue. The primary reason for using PRP is its ability to induce cell proliferation, leading to the accumulation of cells and the development of a robust tissue similar to the original. In addition to promoting cell proliferation, PRP influences the expression of genes in cells, indicating the maturation of tissue and organoid systems. Research has demonstrated that the use of PRP can enhance cell density and maturation by up to 1.5 times compared to conditions without PRP. Furthermore, PRP has been shown to increase the expression of cartilage-related genes and support the maturation of cartilage systems by a factor of ten (Zhang et al. 2017).

Facilitating repair and regeneration studies

Understanding the mechanisms of cartilage repair and regeneration in regenerative medicine is essential for effectively restoring the function of the original tissue. The application of platelet-rich plasma (PRP) can significantly aid in studying cartilage repair mechanisms under controlled conditions. The presence of cartilage organoids in a PRP environment facilitates the investigation of cell repair, migration, proliferation, and extracellular matrix production. The combination of organoids and PRP presents a promising approach for therapeutic purposes and optimizing treatment protocols for cartilage diseases (Xie et al. 2014).

Platelet-rich plasma (PRP) is a mixture of growth factors and inflammatory cytokines that holds great potential for cartilage repair. Since organoid cells can originate from mature cells such as chondrocytes or stem cells, the incorporation of PRP at the center of organoids enhances both cell proliferation and extracellular matrix secretion in the mesenchyme. In preclinical studies, PRP has been utilized in various ways to treat cartilage defects (Fortier and Hackett 2011).

Limitations associated with the integration of PRP into cartilage organoid models

The combination of platelet-rich plasma (PRP) and organoids for treating musculoskeletal and cartilage system diseases represents a novel and promising approach in tissue engineering and regenerative medicine. However, due to its innovative nature and many unknowns, this method presents several challenges and limitations, which are outlined below:

Diversity and variability of PRP composition

Several factors can influence the composition of platelet-rich plasma (PRP), ultimately affecting the structure of the prepared organoids. These factors can be categorized into three main areas:

1. Participant characteristics: Factors such as age, gender, and genetics of the individuals donating blood can significantly impact the quality and composition of the PRP.

2. Storage and maintenance: Conditions such as storage temperature and the duration of plasma storage can lead to alterations in PRP’s properties, potentially diminishing its effectiveness.

3. Preparation methods and protocols: The specific techniques and protocols used in PRP preparation can also vary, leading to differences in composition and efficacy (Tey et al. 2022).

Standardization issues

The absence of a national and standardized protocol for preparing platelet-rich plasma (PRP) with consistent compounds poses significant challenges. As a result, it becomes difficult to predict and ensure uniform responses in PRP treatments and their effects on organoids (Ramaswamy Reddy et al. 2018).

Complex relationship and impact with the organoid environment

To date, there is no comprehensive understanding of the relationship and interaction between platelet-rich plasma (PRP) and organoids. The compounds in PRP can influence the proliferation of cartilage cells, as well as the differentiation and synthesis of their extracellular matrix, depending on variations in PRP composition. Additionally, these compounds may have negative effects on fibrosis and the function of endothelial cells (Yang et al. 2023).

Long-term stability and integrity of organoid in the PRP

Another challenge with cartilage organoids is the limited understanding of how long they remain in host tissue when exposed to platelet-rich plasma (PRP). Over time, the concentration of effective compounds in PRP may decrease, which can impact the efficacy and influence of the organoid. Additionally, it is crucial to determine whether organoids can integrate with the surrounding tissue and maintain their presence in the presence of PRP (Georgakopoulos et al. 2020).

Regulatory and ethical issues

To utilize treatments based on organoids, ethical approval and assurances of safety are necessary. However, proving their safety can be challenging, as the mechanisms underlying their use are not yet fully understood. Additionally, the application of platelet-rich plasma (PRP) necessitates obtaining informed consent from donors (Murray et al. 2022; Mollaki 2021).

Potential of PRP in enhancing the regenerative capacity of cartilage organoids

Platelet-Rich Plasma (PRP) has shown substantial potential in enhancing the regenerative capacity of cartilage organoids, representing a significant breakthrough in tissue engineering and regenerative medicine. PRP is a concentrated source of autologous platelets that are rich in growth factors, including platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-β), and vascular endothelial growth factor (VEGF), among others. These growth factors play crucial roles in cell proliferation, differentiation, and tissue repair (Lana et al. 2023).

When applied to cartilage organoids, PRP significantly enhances their regenerative properties. These three-dimensional constructs closely mimic the structural and functional characteristics of native cartilage and benefit from the bioactive components of PRP. The introduction of PRP into the culture environment promotes enhanced chondrogenesis, the process through which cartilage cells (chondrocytes) form new cartilage tissue. This enhancement is achieved by stimulating chondrocyte proliferation and differentiation, leading to increased production of extracellular matrix components such as collagen and proteoglycans, which are essential for cartilage structure and function (Wang et al. 2023).

Moreover, PRP has been shown to improve the viability and longevity of cells within organoids. The growth factors in PRP can reduce apoptosis (cell death) and enhance the metabolic activity of chondrocytes, creating a more favorable environment for tissue regeneration. Additionally, the anti-inflammatory properties of PRP help mitigate inflammatory responses that can impede the healing process, further supporting the growth and maturation of cartilage tissue (Bacevich et al. 2024).

The application of PRP in cartilage organoids not only accelerates their development but also enhances the quality of the engineered tissue, making it more akin to native cartilage in terms of biomechanical properties and functionality. This improved regenerative capacity is particularly significant for clinical applications, offering a promising strategy for treating cartilage injuries and degenerative diseases such as osteoarthritis. By harnessing the regenerative potential of PRP, researchers and clinicians can develop more effective and reliable therapies for cartilage repair, ultimately improving patient outcomes and quality of life (Thorp et al. 2021).

Cell signaling pathways in cartilage maturation under the influence of PRP

Platelet-rich plasma (PRP) contains growth factors and cytokines that activate various signaling pathways involved in chondrocyte proliferation, extracellular matrix (ECM) synthesis, and cartilage homeostasis. These pathways play a crucial role in cartilage maturation and protection against degeneration. The major signaling pathways influenced by PRP include (Fig. 3):

Fig. 3.

The TGF-β/SMAD signaling pathway plays a crucial role in cartilage regeneration by activating TGF-βRI and TGF-βRII receptors, leading to SMAD2/3 phosphorylation. This enhances the expression of SOX9, COL2A1, and ACAN, essential for chondrogenic differentiation and cartilage maintenance while preventing chondrocyte hypertrophy and ossification. The PI3K/Akt pathway, stimulated by IGF-1 and PDGF in PRP, promotes cell survival, ECM production, and MSC differentiation into chondrocytes by inhibiting GSK-3β and stabilizing β-catenin. The MAPK/ERK pathway, activated by FGF-2 and EGF, regulates chondrocyte proliferation and differentiation, but excessive ERK signaling may lead to hypertrophy and cartilage degradation. The Wnt/β-catenin pathway, influenced by Wnt3a, is essential for cartilage development and ECM synthesis, though its overactivation can induce hypertrophy and matrix calcification. PRP-derived TGF-β helps modulate this balance. Finally, since cartilage is an avascular tissue, HIF-1α signaling is critical for chondrocyte survival under hypoxic conditions. PRP enhances HIF-1α expression, which upregulates VEGF and GLUT-1, supporting cellular adaptation and ECM homeostasis

TGF-β/SMAD signaling pathway

• TGF-β, a key growth factor in PRP, activates TGF-βRI and TGF-βRII receptors, leading to the phosphorylation of SMAD2/3.

• This activation enhances the expression of SOX9, type II collagen (COL2A1), and aggrecan (ACAN), which are essential for chondrogenic differentiation and cartilage maintenance.

• Additionally, TGF-β signaling prevents chondrocyte hypertrophy and ossification, ensuring cartilage stability (Kim et al. 2022).

PI3K/Akt signaling pathway

• Growth factors such as IGF-1 and PDGF, present in PRP, stimulate tyrosine kinase receptors, triggering the PI3K/Akt pathway.

• This pathway promotes cell survival, reduces apoptosis, and enhances ECM production, contributing to cartilage maturation and regeneration.

• Furthermore, Akt inhibits GSK-3β, stabilizing β-catenin, which supports MSC differentiation into chondrocytes and proteoglycan synthesis (Zhang et al. 2018).

MAPK/ERK signaling pathway

• Growth factors like FGF-2 and EGF activate the MAPK/ERK pathway, which regulates chondrocyte proliferation and differentiation.

• However, excessive ERK activation may lead to chondrocyte hypertrophy and cartilage degradation, highlighting the importance of pathway modulation for balanced cartilage development (Wang et al. 2022).

Wnt/β-catenin signaling pathway

• PRP contains Wnt3a and other activators of the Wnt pathway, which bind to Frizzled receptors, leading to β-catenin stabilization.

• This pathway is crucial for cartilage development and ECM synthesis but, when overactivated, can induce hypertrophy and matrix calcification. PRP-derived TGF-β helps regulate this balance (Xia et al. 2020).

HIF-1α signaling in response to hypoxia

• Since cartilage is an avascular tissue, PRP enhances HIF-1α expression, improving chondrocyte survival under hypoxic conditions.

• HIF-1α upregulates VEGF and GLUT-1, supporting cellular adaptation to hypoxia and ECM homeostasis (Jewell et al. 2001).

Clinical implications and future directions

The integration of PRP with cartilage organoid models has profound clinical implications. This combination can lead to more effective treatments for cartilage-related conditions, such as osteoarthritis, traumatic injuries, and other degenerative diseases. Utilizing PRP alongside cartilage organoids could facilitate the development of personalized regenerative therapies, where patient-derived cells and PRP are employed to create custom tissue constructs for implantation. This approach not only enhances the healing process but also reduces the risk of immune rejection and other complications (Xie et al. 2014).

Future research should prioritize optimizing protocols for PRP application in cartilage organoid cultures, focusing on determining the ideal concentration, timing, and delivery methods. Moreover, further studies are essential to uncover the precise molecular mechanisms by which PRP enhances chondrogenesis, extracellular matrix (ECM) production, and cell viability. Understanding these mechanisms will be crucial for refining PRP use in tissue engineering and maximizing its therapeutic potential (Zeng et al. 2023).

Challenges of scaling and hypoxia in organoids

Introduction

Organoids are three-dimensional cell models derived from stem cells that can mimic the structural and functional features of human organs. These models are widely used in biomedical research, drug discovery, disease modeling, and regenerative medicine. However, several challenges, including scaling (Scaling) and hypoxia (Hypoxia), limit the broad clinical applications of this technology. Scaling refers to the ability to mass-produce organoids while maintaining their functional characteristics, whereas hypoxia pertains to the lack of oxygen in the inner regions of organoids, which can lead to cellular necrosis and functional impairment.

In this article, we explore the issues associated with these challenges and analyze the novel solutions proposed in this field.

Challenges of scaling in organoids

Limitations in mass production of organoids

One of the most significant obstacles in the widespread use of organoids is the difficulty in producing large numbers of these structures in a uniform and stable manner. These limitations include:

• Dependence on environmental factors: Organoid formation is influenced by various factors, including extracellular matrix composition, cell density, and culture conditions, leading to heterogeneity in their characteristics (Velasco et al. 2020b).

• Lack of uniformity in size and morphology: Increasing the size of organoids can result in heterogeneous changes in gene expression and cell morphology, reducing their clinical applicability (Gholipourmalekabadi et al. 2016).

• Manual and time-consuming processes: The production of organoids typically requires manual intervention and complex procedures such as cell isolation, culture medium changes, and three-dimensional culture, making mass production challenging (Arjmand et al. 2023).

Role of bioengineering in enhancing scalability

Several strategies have been proposed to increase the scalability of organoids:

• Use of bioreactors: Rotating and dynamic bioreactors can create uniform distribution of nutrients and oxygen, enabling the production of homogeneous organoids (Arjmand et al. 2023).

• Automation of processes: Robotic and AI-based systems can optimize the organoid culture process and facilitate their large-scale production (Kim et al. 2020).

The issue of hypoxia in organoids

Effects of hypoxia on organoids

Hypoxia is one of the fundamental challenges in three-dimensional cell culture and organoids, resulting from the limited penetration of oxygen into the inner regions of these structures. The consequences of this issue include:

• Increased cellular stress and necrosis: Inner cells of organoids experience oxidative stress due to oxygen deficiency, leading to cell death (Malda et al. 2007).

• Activation of hypoxia-related molecular pathways: Oxygen deficiency triggers the activation of hypoxia-inducible factor (HIF-1α), affecting cell differentiation and function (Wörsdörfer et al. 2020).

• Reduced efficiency in disease modeling and drug testing: Hypoxia can alter the physiological characteristics of organoids, decreasing the accuracy of disease models and drug testing.

Strategies to mitigate hypoxia in organoids

Several approaches have been proposed to reduce the effects of hypoxia and improve oxygen delivery in organoids:

• Creation of vascular networks in organoids: Inducing the differentiation of endothelial cells and using angiogenic factors like VEGF can promote the formation of vascular networks and enhance oxygenation (Wörsdörfer et al. 2020).

• Use of oxygen carriers: Calcium peroxide nanoparticles and oxygen-releasing hydrogels provide an innovative strategy for oxygen delivery to the core of organoids (Gholipourmalekabadi et al. 2016).

• Optimization of culture conditions: Increasing dissolved oxygen in the culture medium and using hyperbaric conditions can help improve oxygenation (Ehsan and George 2013).

Conclusion

The integration of Platelet-Rich Plasma (PRP) with cartilage organoids holds immense promise for advancing cartilage tissue engineering. PRP’s ability to enhance chondrogenesis, cell viability, and extracellular matrix production positions it as a powerful tool in developing novel regenerative therapies.

Further research into optimizing PRP application protocols and understanding the underlying mechanisms will be crucial for translating this potential into clinical practice. The potential of PRP to boost the regenerative capacity of cartilage organoids represents a significant advancement in tissue engineering. Enriched with a myriad of growth factors and bioactive molecules, PRP can profoundly influence the cellular environment within cartilage organoids. This enhancement leads to improved chondrogenesis, accelerated cellular proliferation, and increased extracellular matrix production, which are critical for developing and maturing functional cartilage tissue.

Overall, the integration of PRP with cartilage organoids not only enhances their regenerative potential but also provides a promising avenue for effective treatments for cartilage injuries and degenerative diseases, offering a transformative impact on clinical cartilage repair and restoration strategies.

Abbreviations

ECM

Extracellular matrix

PDGF

Platelet-derived growth factor

FGF

Fibroblast growth factors

HSPG

Heparan sulfate and proteoglycan

TNF

Tumor necrosis factor receptor

BMP-2

Bone morphogenetic protein-2

VEGF

Vascular endothelial growth factor

IL4

Interleukin 4

DEX

Dexamethasone

NPs

Nanoparticles

µM

Micromolar

mM

Milli molar

nM

Nanomolar

mg/ml

Mili gram/mile liter

Author contributions

MG, HD; Conceptualization, Methodology, MP; Data curation, Writing- Original draft preparation, AR, Supervision. MR; Writing, Reviewing ZO, Editing. All authors read and approved the final manuscript.

Funding

There is no financial source to support the article and it is not provided by any organization.

Data availability

The data that support the findings in this study will be shared on reasonable request to the corresponding author.

Declarations

Competing interests

The authors declare that they have no competing interests.

Ethical approval

We did not receive informed consent or confirmation of the patient study because it was a review study without patient intervention.

Consent for publication

All authors are fully satisfied with the publication of the article.