Nucleic acid hybridization is a key molecular biology technique for detecting and analyzing specific DNA or RNA sequences based on base complementarity. Among its methods, the Southern blot stands out, involving DNA extraction, restriction enzyme digestion, electrophoretic separation, and transfer to a membrane for hybridization with a labeled DNA probe [30–32]. The Northern blot follows a similar approach but targets RNA [33, 34]. A significant variant is in situ hybridization (ISH), performed directly on tissues, cells, or chromosomes, first applied in 1969 to detect DNA in Xenopus. This technique has since advanced into a crucial tool for studying gene expression, cell proliferation, apoptosis, and disease-related genes, including cancer-related ones [35]. Its RNA application focuses on detecting specific mRNAs. Hybridization relies on the renaturation of single-stranded DNA or RNA following denaturation, which disrupts hydrogen bonds and hydrophobic interactions through heat or chemical agents. Controlled cooling allows complementary strands to reanneal, forming hybrids if the pairing strand differs from the original. This property enables hybridization-based gene and nucleic acid identification [36]. When talking of ISH, two key components are required: A target sequence generated by proteolytic enzymes and a labeled probe complementary to it. The probe, marked with a radioactive, fluorescent, or enzymatic tag, enables visualization of hybridization sites within tissues or cells [36]. In biomaterials research, ISH serves as a crucial tool for assessing biocompatibility, offering molecular insights into material-tissue interactions, and validating biomedical applications.

Below is a simplified protocol for implementing ISH, which is graphically summarized in Figure 2. For the complete process, refer to [37, 38]. In Fluorescent in situ hybridization (FISH), the preparation and application of probes are critical for accurate detection of chromosomal abnormalities. Three main types of probes are commonly used: chromosome painting probes, which label entire chromosomes and enable the identification of structural and numerical rearrangements using techniques like Multicolor FISH (M-FISH) and Spectral Karyotyping (SKY); repetitive sequence probes, which target repetitive chromosomal regions such as telomeric or centromeric sequences, aiding in the detection of aneuploidies like monosomies or trisomies; and locus-specific probes, which are derived from genomic clones in PAC or BAC vectors and are used to detect specific chromosomal aberrations such as translocations, deletions, or inversions.

Cytological preparation is essential for clear hybridization signals (Figure 2a). For chromosomal studies, cells—such as root tips in the mitotic phase—are fixed in an ethanol:acetic acid solution (3:1), stained with 1% acetocarmine, squashed in 45% acetic acid, and can be stored at –80°C for up to a year. DNA probes are labeled (Figure 2b) using non-radioactive molecules like biotin, digoxigenin, fluorescein, or rhodamine through techniques such as nick translation, random priming, or PCR. Detection is then performed using antibodies conjugated to fluorochromes or enzymes.

ISH methods (Figure 2c–g) vary depending on the approach. In non-radioactive ISH, chromosomal DNA is denatured with formamide and SSC at 68–70°C, the probe is applied and incubated, and hybridization sites are detected with streptavidin-peroxidase and developed with DAB (to be defined) and H₂O₂, followed by Giemsa counterstaining. Genomic in situ hybridization (GISH) involves fragmenting genomic DNA, labeling it with biotin-16-dUTP, and hybridizing similarly, using the same detection steps. In FISH, the DNA is denatured, the fluorescently labeled probe is hybridized, and the slides are blocked with PBS and Triton-100, then incubated with fluorescent antibodies (e.g., FITC). A counterstain with propidium iodide allows the observation of fluorescent signals under a microscope, enabling precise chromosomal analysis.

ISH can be used to investigate how biomaterials interact with cells in a living environment. This tool is valuable for understanding cellular responses to implanted materials and how they interact with the genetic material of surrounding cells. In addition to reflecting the genetic interaction between biomaterials and host cells, ISH also allows for the evaluation of gene expression, immune response, and other aspects related to material biocompatibility. Some of the earliest applications of this technique in this field date back to 1996, when the Institute of Pathology and the Department of Trauma and Reconstructive Surgery in Germany analyzed osteoblast activity at the biomaterial-bone interface using ISH. In this study, the amount of mRNA expressed by osteoblasts was evaluated, confirming RNA loss during the decalcification process [39].

Additionally, ISH has a direct application in biomaterial development. One example is a study that proposed a new method for the biosynthesis of a three-dimensional scaffold using the in-situ hybridization of carbon nanotubes (CNT) with bacterial cellulose (BC). This method achieved homogeneous and effective hybridization between CNT and BC, resulting in hybrid scaffolds with excellent osteoinductive and osteoconductive properties. This, in turn, promoted higher bone density, improving regeneration efficiency. This innovation highlights the potential of ISH of three-dimensional scaffolds with nanomaterials as a new approach for regenerative medicine [40].

To provide a clearer and more structured overview of ISH applications in biomaterials research, Table 1 summarizes key case studies from the literature. These examples demonstrate how ISH has been used to assess biocompatibility, immune responses, and scaffold performance by targeting specific genes or cellular processes.

However, the ISH technique faces limitations, such as difficulty in identifying targets with low DNA or RNA copy numbers. Despite this, strategies continue to be developed to improve its sensitivity, either by amplifying nucleic acid sequences before hybridization or by enhancing signal detection after completion [43]. In a study evaluating gene expression changes on biomaterial surfaces, the combination of ISH for mRNA extraction with real-time quantitative PCR allowed researchers to identify expressed genes. However, this technique did not provide information on the abundance of different cell types, which is problematic since inflammatory processes mainly involve neutrophils and macrophages in wound healing. Understanding the balance between these cells and the presence of specific macrophage subpopulations is crucial for a better comprehension of these processes [44].

Although there are areas requiring further development, the information obtained so far is fundamental to ensuring the safety and advancement of any material intended for biological applications. ISH is used to study the effects of biomaterials on the gene expression of exposed cells, allowing researchers to observe how materials influence the activation or repression of key genes for biocompatibility. For example, ISH can assess the expression of genes related to inflammation or cell proliferation in tissues interacting with implants or scaffolds.

Genetic engineering has led to the development of recombinant DNA, a groundbreaking tool in modern molecular biology. By combining DNA sequences from different organisms, recombinant DNA allows for precise genetic manipulation, enabling the modification of traits and the introduction of new biological functions. This technology has revolutionized fields like medicine, agriculture, and biomaterials, facilitating advances in biotechnology with high precision and control [45]. Central to recombinant DNA technology are restriction enzymes, which cut DNA at specific sites, and DNA ligase, which joins the fragments to form new molecules. These recombinant molecules are introduced into host organisms, such as bacteria, yeast, or mammalian cells, where they can replicate and express proteins or molecules. This technique has enabled the large-scale production of human proteins, including insulin, growth factors, and monoclonal antibodies [45, 46].

In biomaterials, recombinant DNA technology offers new possibilities for designing functional materials. It allows for the modification of biological and physical properties, optimizing interactions with cellular and tissue environments. By introducing specific genes into cells interacting with biomaterials, researchers can control protein production to promote tissue regeneration, wound healing, or implant integration [47, 48]. A key application of recombinant DNA in biomaterials is creating platforms for controlled expression of bioactive molecules. For example, tissue engineering scaffolds can be designed to contain genetically modified cells that secrete growth factors, enhancing tissue formation. This is especially important in bone and cartilage regeneration, where proteins like BMP (bone morphogenetic protein) are essential for cellular differentiation and bone formation [49].

Recombinant DNA technology also plays a vital role in improving biomaterial biocompatibility and reducing immune system rejection. By incorporating genes that encode immunomodulatory molecules, biomaterials can be designed to regulate immune responses, minimizing inflammation and rejection in applications like stents, prostheses, and other medical implants [47, 48]. Additionally, recombinant DNA enables the development of responsive biomaterials. By incorporating genes that react to external stimuli like temperature, pH, or mechanical signals, biomaterials can adjust their properties in real-time to meet the needs of the biological environment. For example, biomaterials that release therapeutic proteins in response to inflammation could be used to treat conditions like rheumatoid arthritis or cancer, expanding the potential of recombinant DNA in personalized medicine [49].

Ultimately, recombinant DNA technology is not only advancing biomaterials but also enabling the creation of more accurate models for human diseases, aiding the development of new therapies. In summary, recombinant DNA has ushered in a new era in biomaterials science, blending biology and engineering to create materials that are not only compatible but actively involved in healing and regeneration processes [47].

This section outlines a generic protocol for applying recombinant DNA technology to biomaterials, which is graphically summarized in Figure 3. For a more detailed overview of the protocol, refer to references [50, 51]. The process of creating recombinant DNA begins with the selection of appropriate vectors and host organisms (Figure 3a), where the host is chosen based on the intended biomedical application, and the cloning vector is a DNA molecule capable of replicating within the host while carrying the target gene. Next, both the vector and the target DNA are prepared (Figure 3b, c) by isolating them and using restriction enzymes to cut at specific sites, allowing for precise ligation. The recombinant DNA is then formed (Figure 3d) by combining the prepared vector and target DNA using DNA ligase, resulting in a single construct containing the desired gene. This recombinant DNA is introduced into the host organism through transformation methods such as electroporation or the use of viral vectors (Figure 3e), followed by the selection of host cells that successfully incorporate the recombinant DNA. Finally, detection and identification are carried out (Figure 3f) using techniques like PCR, restriction analysis, or sequencing to confirm successful incorporation, after which the recombinant DNA is expressed.

Recombinant DNA technology is a fundamental tool in modern biotechnology, used to produce key biomolecules, such as therapeutic proteins, which are utilized in the creation of biomaterials for various medical interventions. Through genetic engineering, it is possible to insert genes of interest into host organisms such as bacteria, yeasts, or mammalian cells, enabling them to produce proteins that would otherwise be difficult or costly to obtain. Clear examples include the production of human insulin, growth hormones, and clotting factors, which have revolutionized the treatment of diseases such as diabetes and hemophilia. These recombinant proteins can be integrated into biomaterials to enhance their functionality in medical applications, such as scaffolds for tissue regeneration or matrices for controlled drug delivery [51].

The first breakthrough in medical biotechnology using recombinant DNA technology was the production of human insulin by Genentech in the 1980s. By inserting the human insulin gene into Escherichia coli bacteria, a safe and efficient source of insulin for diabetes treatment was achieved. This recombinant insulin eliminated the dependency on animal-derived insulin, which carried the risk of adverse immune reactions [53]. In hemophilia A, a deficiency in coagulation factor VIII causes severe blood clotting issues. Recombinant DNA technology enabled the production of factor VIII in mammalian cells, offering a safer and purer alternative to human plasma concentrates, which carried the risk of transmitting viruses such as HIV and hepatitis [54]. This treatment revolutionized the lives of hemophilia patients by providing reliable therapy with no risk of infection.

Collagen is a structural protein essential in tissues like skin, tendons, and bones. The production of recombinant human collagen has been achieved by inserting collagen genes into yeast or mammalian cells. A notable example is the company FibroGen, which produces recombinant collagen for use in manufacturing dermal patches used in the healing of chronic wounds such as diabetic ulcers, offering a safer alternative to animal-derived collagen [55, 56]. In the field of regenerative medicine, genetically modified stem cells have been used to enhance tissue regeneration. An example is the use of mesenchymal stem cells genetically modified to express specific growth factors, such as transforming growth factor beta (TGF-β), which is crucial in bone tissue repair. These cells, combined with biodegradable biomaterial scaffolds, have demonstrated accelerated bone fracture healing in clinical trials [57, 58]. Recombinant collagen has been applied as hydrogels in combination with other biomolecules, such as chitosan (Figure 3g), obtaining that the recombinant collagen presence improves several aspects of this biomaterial, such as the biocompatibility of these hydrogels, cell infiltration, and accelerates wound healing [52].

Recombinant DNA technology has also benefited the development of monoclonal antibodies [59]. Monoclonal antibodies, such as trastuzumab (Herceptin), are recombinant proteins produced using recombinant DNA technology and are used to treat cancers like HER2-positive breast cancer. These antibodies specifically target cancer cells expressing the HER2 receptor, blocking their growth and signaling. Recombinant DNA technology has enabled the large-scale production of these antibodies, improving the treatment of aggressive cancers [60]. An emerging application is the controlled release of recombinant proteins through biomaterial matrices. A notable case is the development of a controlled release system for recombinant VEGF (vascular endothelial growth factor) to promote angiogenesis in tissue regeneration. This system, based on biodegradable polymers, has shown effectiveness in treating ischemic wounds by promoting the formation of new blood vessels [61, 62]. These key examples of recombinant DNA applications in regenerative and therapeutic medicine (where therapeutic medicine refers to medical approaches aimed at treating or curing diseases) are summarized in Table 2, highlighting the host systems, medical uses, and associated benefits of each strategy.

PCR is a fundamental technique in molecular biology that enables the amplification of specific DNA sequences from minimal samples. Developed by Kary Mullis in the 1980s, PCR has transformed genetic analysis, disease diagnostics, and biotechnology by facilitating the exponential replication of target DNA regions [67]. The process involves cyclic thermocycling steps: denaturation (separating DNA strands), primer annealing (binding of short DNA primers), and extension (DNA polymerase-driven synthesis of new strands). Essential reaction components include template DNA, primers, nucleotides (dNTPs), DNA polymerase, buffer, and Mg²⁺ ions. The amplified DNA can be analyzed using agarose gel electrophoresis to confirm successful amplification [68–70].

PCR plays a crucial role in biomaterial research by enabling the characterization of genetic material extracted from biological samples. It is used to identify genetic variants, analyze gene expressions, and investigate critical molecular interactions in fields such as tissue engineering and biomedical diagnostics [71–73]. Additionally, real-time PCR (RT-PCR) allows the study of RNA by converting it into complementary DNA (cDNA) via reverse transcription [67, 68].

This method, which is both rapid and highly efficient, has revolutionized areas like genetic research, molecular diagnostics, and the development of targeted therapies, among other biotechnological applications. PCR is a method that allows for the exponential amplification of DNA sequences. It uses a heat-stable DNA polymerase to replicate specific DNA segments through controlled temperature cycles. This technique can also be adapted to RT-PCR to work with RNA, converting it to cDNA using the enzyme reverse transcriptase.

This section outlines a generic PCR protocol with specific applications in biomaterials research, which is graphically summarized in Figure 4. PCR follows a series of essential steps to ensure successful DNA amplification. These steps include primer design, reaction mixture preparation, PCR execution, and thermal cycling adjustments. For a more exhaustive description of the procedure, it is recommended to consult references [74, 75].

PCR is a key tool for assessing gene expression in response to biomaterial interactions with living tissues. It is widely used to study genes involved in inflammatory responses, wound healing, and tissue regeneration, providing insights into the biocompatibility of biomaterials.

Biomaterials, whether natural or synthetic, are designed for medical applications to restore or replace biological functions. Despite their promising physical, chemical, and biological properties, they do not always integrate seamlessly with living tissues. A common issue is the immune system recognizing the material as foreign, triggering an inflammatory response. PCR allows for the identification of gene expression changes, helping to evaluate tissue reactions at the molecular level (Table 5 summarizes representative examples of these applications in various tissues and biomaterials).

PCR has been applied to study biomaterial-tissue interactions in various contexts. For instance, biomaterial patches in the gastrointestinal tract were analyzed, revealing increased expression of the vitamin D binding protein in gastric tissue following implantation [79]. Similarly, in studies of bioactive polymer scaffolds for bone regeneration, PCR has been used to assess osteogenesis-related genes, demonstrating how biomaterials influence stem cell differentiation into osteoblasts [80]. In cardiac tissue engineering, PCR has helped monitor the gene expression in biomaterial grafts that simulate myocardium, identifying materials that promote integration without chronic inflammation [81, 82]. Even this technique has been helpful to determine if modified collagen hydrogels can modulate the inflammatory response, promote regeneration in ex vivo intervertebral discs rupture models, by quantifying the expression of genes that code inflammatory cytokines IL-6 and iNOS, and the extracellular matrix protein aggrecan (Figure 4h) [24].

In research on biocompatibility, PCR has been used to evaluate inflammatory markers such as IL-6 and TNF-α in macrophages interacting with biomaterials, providing critical data for optimizing material design [83, 84]. PCR has also been employed to study angiogenesis by analyzing VEGF expression in tissues implanted with bioactive scaffolds, which is essential for applications requiring vascularization, such as chronic wound healing [85]. Additionally, in nerve regeneration studies, PCR has been used to measure the expression of NGF and BDNF, which are crucial for neuronal survival and repair, helping identify biomaterials that support nerve growth [86].

In bone regeneration, PCR has been applied to analyze gene expressions in response to materials like hydroxyapatite and bioglass, focusing on markers such as RUNX2 and ALP, which are essential for osteointegration [87]. Understanding the molecular response to biomaterials is fundamental for improving their design and ensuring their functionality without triggering adverse biological reactions.

Despite its advantages, PCR has limitations. Sample preparation is critical for accurate results, and conventional PCR requires complementary techniques like gel electrophoresis for interpretation. RT-PCR enables precise quantification but requires specialized equipment and strict sample handling. Additionally, the technique is limited in amplifying long DNA sequences, which can be a constraint in complex biomaterial studies [88, 89].

PCR remains an essential technique for evaluating biomaterial tissue interactions, providing valuable information for the development of more effective and biocompatible medical materials.

ICC is a powerful technique for visualizing cell proteins using labeled antibodies. Developed in the 1940s by A.H. Coons, it has since evolved into a crucial method in histopathology, cytopathology, and biomedical research. ICC can be applied to both cultured cells and tissue sections using fluorescence or visible light microscopy, depending on the marker [90, 91]. A key feature of ICC is its ability to be used in both animal and plant cells, provided a specific antibody is available. This technique relies on antigen-antibody interactions, allowing precise protein localization. The specificity of antibodies enables the detection of minimal antigen amounts, making ICC a highly sensitive method. Advances in antibody generation continue to expand its applications [92, 93]. Initially, ICC was limited to cultured cells, while IHC was reserved for paraffin-embedded tissues. Today, ICC is widely used for formaldehyde-fixed animal tissues, which can be cryosectioned and incubated with antibodies [94].

The core principle of ICC is the antigen-antibody reaction. Antibodies, produced by the immune system in response to antigens, can be purified and conjugated with fluorescent dyes for detection. ICC employs two types: polyclonal antibodies, which recognize multiple epitopes of an antigen, and monoclonal antibodies, which bind to a single epitope [95, 96]. For instance, a polyclonal antibody against actin can be generated by injecting purified actin into a rabbit, triggering an immune response. The resulting antibodies are then isolated and labeled for use in ICC [97]. ICC techniques include: i) Direct Immunocytochemistry: Uses a primary antibody directly conjugated to a fluorophore or enzyme. While simple, it has limited sensitivity [98]. ii) Indirect Immunocytochemistry: Enhances sensitivity by using an unlabeled primary antibody, followed by a labeled secondary antibody that binds to the primary. This amplification improves detection and reduces the need for labeling multiple primary antibodies individually [99].

Detection markers vary depending on the application. Fluorescent dyes allow imaging with fluorescence or confocal microscopy, enzymatic markers like peroxidase enable colorimetric detection under a light microscope, and colloidal gold nanoparticles are used in electron microscopy [100]. ICC is extensively used in oncology for detecting tumor biomarkers, as well as in basic research to study protein distribution in cells and tissues. It has been instrumental in understanding processes such as the cell cycle, apoptosis, and intracellular signaling [101–103].

This section outlines a standardized protocol for ICC with specific applications in biomaterials research, which is graphically summarized in Figure 5. For a more detailed procedure, refer to references [104–106].

ICC enables the analysis of cellular interactions with biomaterials by identifying key proteins involved in adhesion, proliferation, and differentiation. These techniques have revolutionized biomaterials research by providing detailed insights into the physiological responses triggered by implants [108–110]. Initially, biomaterials were designed to minimize immune responses and avoid rejection or chronic inflammation. However, advances in regenerative medicine have shifted the focus toward bioactive biomaterials capable of modulating cellular responses to promote tissue regeneration [1–3].

For instance, in bone regeneration, biomaterials should not only be biocompatible but also stimulate osteoblast activity. Here, immunochemical techniques play a crucial role by enabling the analysis of these complex interactions, providing detailed information on how cells respond to different implanted materials [111, 112]. Immunocytochemical studies have evaluated titanium surfaces for cell adhesion, revealing that differentiated osteoblasts respond favorably to surface roughness while stem cells proliferate independently of topography [108]. This type of analysis is essential for developing optimized surfaces for medical implants, as cell adhesion and proliferation are determinants of implant success. Similarly, collagen matrices for urethral reconstruction in rabbits demonstrated proper integration and muscle fiber organization six months post-implantation [113]. Hydroxyapatite implants have been analyzed using IHC, revealing increased expression of osteopontin and TGF-β, key markers of osteogenesis [114]. In vascular engineering, electrospun polymer grafts assessed with endothelial markers (CD31, vWF) demonstrated new blood vessel formation and cellular integration in rat models, while recent advances in microvascular tissue engineering explore biofabrication strategies such as extrusion-based and droplet-based bioprinting, Kenzan, and biogripper techniques to create nano- and micro-sized aggregates and microspheres that mimic capillary networks through the inclusion of specific cell types and proangiogenic factors [115, 116].

Collagen scaffolds modified with silver nanoparticles (AgNPs) have been shown to reduce proinflammatory macrophages (CD68) and increase reparative macrophages (CD206), creating a favorable environment for wound healing [117]. Additionally, cross-linked collagen hydrogels either containing colloidal silica or modified with epoxyeicosatrienoic acids promoted an anti-inflammatory macrophage response, identified via CD206 immunostaining [14, 24, 25, 118]. The immunomodulatory effect of plain silicon nanoparticles has been evaluated, where immunocytofluorescence has been used to prove that the inflammatory response of macrophages to these particles can be dose-regulated, proving that doses over 40 μg/mL promote the expression of the proinflammatory cytokine iNOS (Figure 5e), while lower doses promote the expression of the antiinflammatory molecules CD206 and TLR1 [107].

Myocardial patches using induced pluripotent stem cells (iPSCs) on fibrin gel scaffolds have been evaluated through immunohistochemical techniques, confirming differentiation into cardiomyocytes via troponin I and α-actinin expression [119, 120]. Controlled drug release systems using biodegradable PLGA polymers have been studied in animal models, with immunohistochemical analysis revealing an initial inflammatory response that was mitigated over time by integrated anti-inflammatory drugs, demonstrating their potential for chronic inflammatory conditions [121, 122].

Despite their advantages, immunochemical techniques have limitations, including potential errors in sample preparation, antibody concentration optimization, and cross-reactivity. Fluorescence-based methods require careful selection of filters and lasers to prevent signal interference [123, 124]. Future developments in 3D bioprinting and tissue engineering will increasingly rely on ICC to evaluate cellular responses to novel biomaterials. The integration of these techniques with super-resolution microscopy and single-cell protein analysis will deepen our understanding of biomaterial-cell interactions, opening new frontiers in regenerative medicine and personalized therapies [104].

IHC is a technique that enables the localization of proteins in tissue sections, allowing for the visualization of molecular structures using enzymatic or fluorescent detection methods [124, 125]. Combining principles from immunology, histology, and chemistry, IHC plays a crucial role in biomaterials research by identifying biological markers related to inflammation, wound healing, angiogenesis, apoptosis, and tissue regeneration [126–133]. Scientific literature provides guides, protocols, and textbooks that are essential for those seeking to deepen their use of IHC and achieve proper preparation for biomaterial evaluations. Biocompatibility is fundamental for medical applications, as it defines a material’s ability to interact with biological systems without adverse effects. IHC is widely used to evaluate biomaterials in both in vivo and in vitro studies, providing insights into immune responses and tissue integration [6, 126].

The technique relies on antibodies, primarily from the IgG class, which specifically bind to target antigens [125, 134]. Detection can be direct, where the primary antibody is conjugated with a marker, or indirect, using a secondary antibody to enhance signal sensitivity [135]. Fluorescent markers are preferred for frozen tissues, while enzymatic labels are used in formalin-fixed, paraffin-embedded samples [136]. Proper tissue fixation is essential for preserving molecular structures. Cross-linking fixatives, such as formaldehyde, stabilize proteins, whereas precipitating fixatives, like alcohols, remove water to preserve tissue morphology [125]. Both types are selective for proteins, while lipids and carbohydrates remain trapped within them.

Fixation methods include perfusion, distributing the fixative through the vasculature, immersion, and submerging tissue in a fixative solution [125]. Samples are then sectioned into thin slices (5–15 µm) for microscopic analysis. Frozen tissue preparation requires precision to prevent ice crystal formation, which can damage cellular structures. A sucrose solution is often used to protect tissue integrity before embedding in a suitable compound for sectioning and analysis [137]. These controlled procedures ensure optimal conditions for IHC, enhancing the accuracy of biomaterial evaluations.

This section describes a general IHC protocol with specific applications in biomaterials research, which is graphically summarized in Figure 6. Refer to references [138, 139] for a more comprehensive description. The IHC process is summarized below.

IHC is a powerful tool for evaluating how biomaterials influence biocompatibility by analyzing inflammatory, angiogenic, and apoptotic markers in surrounding tissues. This technique enables the investigation of tissue-biomaterial interactions, providing insights into specific proteins associated with cellular responses such as adhesion, proliferation, and differentiation within the tissue context. Understanding these molecular-level interactions is crucial for assessing how implanted biomaterials modulate cellular behavior within tissues. By allowing the precise localization of key proteins, IHC has revolutionized biomaterials research, offering a detailed perspective on physiological processes triggered in response to implants [141].

In a study on hyaluronic acid-based hydrogels for IL-10 delivery in idiopathic pulmonary fibrosis (IPF), IHC played a critical role in identifying and quantifying fibrosis-related markers and cellular activity in lung tissue treated with IL-10 hydrogels. Using specific antibodies, IHC detected proteins associated with fibrosis, such as type I collagen and TGF-β, in tissue sections obtained after intratracheal administration of the treatment in a murine model. This allowed for an assessment of IL-10’s antifibrotic effects, demonstrating a reduction in collagen expression and decreased activation of fibroblasts and myofibroblasts in lung tissue. IHC provided detailed histological and molecular insights into treatment-induced changes, confirming the potential of this IL-10 delivery system in mitigating fibrosis progression and preserving lung structure [142].

In a study on calcium carbonate (CaCO₃)-based composite hydrogels for bone regeneration, IHC played a critical role in identifying and quantifying osteoimmunomodulatory markers and cellular activity in bone defects treated with BMP-2 and catalase (CAT)-loaded hydrogels. Using specific antibodies, IHC detected proteins associated with inflammation and bone healing, such as CD80 and CD206, in tissue sections obtained after implantation in an animal model. This allowed for an assessment of the hydrogel’s ability to regulate macrophage polarization, demonstrating that CAT presence in the composites downregulated the expression of CD80 (Figure 6h), and upregulated the expression of CD206, associated with proinflammatory M1, and anti-inflammatory macrophage phenotypes, respectively. Additionally, IHC provided detailed histological and molecular insights into treatment-induced changes, confirming the potential of this biomaterial to modulate the bone microenvironment, enhance osteogenesis, and promote bone defect healing [140].

Similarly, in tissue regeneration studies, IHC was used to evaluate the efficacy of bioactive polypeptide hydrogels (QK-SF) in modulating macrophage polarization and promoting skin regeneration in a murine wound model. IHC enabled the visualization and quantification of macrophage subpopulations in wound tissue, highlighting the shift from proinflammatory M1 macrophages to pro-healing M2 phenotypes. This was evidenced by an increase in M2-specific markers and a reduction in M1 markers in skin sections treated with the hydrogel. Additionally, IHC facilitated the assessment of keratinocyte differentiation and collagen deposition, providing a comprehensive view of tissue healing and regeneration. The findings, supported by in vitro data, reinforced the conclusion that QK-SF hydrogel not only promotes angiogenesis but also regulates immune responses, making it a promising candidate for tissue engineering and wound healing applications [143].

In the context of coronary artery disease treatment, IHC was utilized to assess the effectiveness of an immunomodulatory biomaterial in macrophage polarization and cardiac tissue regeneration following myocardial infarction. IHC analyses of heart tissue sections from rats treated with alginate gels loaded with immunomodulatory factors, such as colony-stimulating factor (CSF-1) and anti-inflammatory interleukins (IL-4/6/13), revealed the expression of markers associated with prohealing macrophages. This indicated a cellular shift toward phenotypes that support tissue repair. Furthermore, IHC was complemented by echocardiography to evaluate overall cardiac function, providing a comprehensive assessment of the treatment’s impact on myocardial recovery. The results underscored the therapeutic potential of the proposed biomaterial, demonstrating its ability to modulate immune responses and enhance cardiac function, suggesting a novel approach for ischemic tissue revascularization [144].

In a study on pelvic organ prolapse (POP), IHC was employed to evaluate the effectiveness of a biologically functionalized hydrogel in promoting connective tissue repair in abdominal wounds in rabbits. IHC analysis of fibroblast regeneration markers in tissue sections obtained after injection of polyisocyanide (PIC) hydrogel provided insights into new tissue growth and immune activity at the hydrogel-tissue interface, indicating a favorable therapeutic response. Additionally, IHC identified cellular composition changes and extracellular matrix remodeling, crucial for understanding how hydrogel injection enhances collagen deposition and tissue stiffness over time compared to controls. These findings support the hypothesis that PIC hydrogel induces a regenerative fibroblast response, offering an innovative solution for improving surgical outcomes in POP patients and motivating further translational research [145].

Decellularized bronchial grafts have been investigated as promising biomaterials for airway reconstruction, addressing a critical clinical need. These grafts, derived from porcine lungs, demonstrated effective integration with native tissue and epithelial layer formation, as evidenced by histological and electron microscopy analyses. IHC revealed proper revascularization, with positive CD31 staining indicating a favorable healing environment. Although a slight reduction in tensile strength was observed after one month, it was restored within two months. The immune response to the grafts was comparable to that of autografts, suggesting that these biomaterials are safe and could be applied clinically for tracheal reconstruction, offering innovative solutions for patients with airway defects [146].

Overall, IHC is an essential tool in biomaterials research, allowing for the assessment of graft-host tissue interactions. This technique provides critical insights into processes such as revascularization, re-epithelialization, and immune responses, offering valuable information on biomaterial efficacy and safety in tissue regeneration. Highlighting its significance underscores the crucial role of IHC in advancing regenerative therapies and its potential to improve clinical outcomes across various biomedical applications.