Design and clinical application of injectable hydrogels for musculoskeletal therapy

Abstract Musculoskeletal defects are an enormous healthcare burden and source of pain and disability for individuals. With an aging population, the proportion of individuals living with these medical indications will increase. Simultaneously, there is pressure on healthcare providers to source efficient solutions, which are cheaper and less invasive than conventional technology. This has led to an increased research focus on hydrogels as highly biocompatible biomaterials that can be delivered through minimally invasive procedures. This review will discuss how hydrogels can be designed for clinical translation, particularly in the context of the new European Medical Device Regulation (MDR). We will then do a deep dive into the clinically used hydrogel solutions that have been commercially approved or have undergone clinical trials in Europe or the United States. We will discuss the therapeutic mechanism and limitations of these products. Due to the vast application areas of hydrogels, this work focuses only on treatments of cartilage, bone, and the nucleus pulposus. Lastly, the main steps toward clinical translation of hydrogels as medical devices are outlined. We suggest a framework for how academics can assist small and medium MedTech enterprises conducting the initial clinical investigation and post‐market clinical follow‐up required in the MDR. It is evident that the successful translation of hydrogels is governed by acquiring high‐quality pre‐clinical and clinical data confirming the device mechanism of action and safety.


| HYDROGELS AS MEDICAL DEVICES
Hydrogels represent a group of biomaterials consisting of waterswollen polymer or colloidal networks. 1 Hydrogels are viscoelastic materials that have attracted attention in regenerative medicine due to their ability to structurally mimic the extracellular matrix (ECM), 2 thereby creating a conducive environment for cell proliferation and tissue regeneration. The viscoelastic properties of hydrogels allow them to function as stem cell carriers or scaffolds for controlled drug release. Within the review by Correa and colleagues, 3  Recently, Catoira et al. 5 have discussed how the regulation affects the translation of hydrogels. Hence, this review will consider how hydrogels can be designed to satisfy these regulations. However, expanding cells or integrating cell-stimulating therapeutics into the medical device results in these hydrogel systems being regulated as medicinal products (drugs/biologics). Indeed, they are considered as drugs when their principal mode of action is pharmacological, metabolic, or immunological. 6 The consequence of the medicinal regulation is that a more thorough investigation of the biocompatibility and therapeutic effect is required before such solutions can be approved for clinical application. This increases the translational barriers and the time before patients can benefit from the treatment. Therefore, it is attractive to translate hydrogels solutions as medical devices such that the therapy can reach the clinic earlier and is more affordable. A detailed discussion of the classification can be found in Section 4.
Particularly in applications for musculoskeletal disorders, there is an unmet need for minimally invasive therapies, where the use of injectable hydrogels has tremendous potential. The demand is driven by an aging population that gives two unique opportunities: (1) an increasing number of patients outlive the longevity of permanent medical devices; thus, hydrogel therapies can be used to delay permanent implantation, (2) minimally invasive therapies give a treatment opportunity for the growing population group that would otherwise not survive the trauma induced by conventional surgeries. 7 Examples of these type of devices are represented by hydrogels for joint lubrication, 8,9 injectable scaffolds for guided bone 10 or cartilage regeneration, 11 or nucleus pulposus (NP) replacements. 12 These are widely different applications for diverse tissues with different loading modes and levels. Consequently, a one-fit-all hydrogel is an unlikely strategy, and thought should be put into the clinical requirements of the material when designing the hydrogel.

| HYDROGEL DESIGN
Hydrogels are an extensively investigated class of biomaterials, and an increasing number of products have reached the clinic. In the following section, we will go through the design steps of the hydrogels and discuss what considerations need to be taken to improve the likelihood of clinical translation and to comply with the European Medical Device Regulations (MDRs). The design process is summarized in

| Material selection
The first step in the design process is to select a suitable polymer to form the hydrogel. There is a larger group of naturally derived polymers such as collagen, 13 hyaluronic acid (HA), 14 chitosan, 15 cellulose, 16 and alginate. 17 Although not exclusively, plant-based polymers tend to be composed of saccharides, such as cellulose, and animal-based polymers tend to compose of protein, for example, collagen. 18 These have been attractive as their natural origin makes them favorably biocompatible and biodegradable but can introduce issues such as immunogenicity and limited mechanical properties. 19 From a translational perspective, these are limited by high cost and batch-tobatch variability. 5,19,20 Alternatively, synthetic polymers such as poly(ethylene glycol) (PEG), 21 poly(vinyl alcohol), 22 poly(acrylic acid), 23 and poly(acrylamide) 24 can be used. Synthetic polymers are industrially more used as they are more favorable from both cost and regulatory perspective, the two being also connected. Synthetic polymers can be produced in more robustly repeatable manners and more efficiently with respect to naturally derived ones, making them readily scalable. 25 Synthesis is typically a more straightforward production process and ensures controlled environmental factors thus limiting the risk of contamination. Synthetic polymers are favorable versus naturally derived raw materials as they allow for improved traceability and higher degree of availability which finally reduces the cost. 25,26 However, their clinical adoption has been limited, and those that exist usually provide a mechanical mechanism of action, for example, PEGhydrogel as a spacer between prostate and rectum to protect the rectum during radiotherapy. 27 For the regenerative market, the translation is insignificant, which has been attributed to their low biocompatibility. 28 The low biocompatibility is likely related to lack of cell-specific bioactivity, including cell adhesive and migratory cues, and cell-mediated material degradation. 29 This highlights how biocompatibility is vital for the success of any hydrogel, and the biological response should be central to the choice of the polymer for the hydrogel. Implanted materials can either integrate physiologically, leading to minimal or no scaring, or the material can induce chronic inflammation and a foreign body response. 30 After injection, the material must provide appropriate biochemical and biophysical signals to recruit host cells that will eventually produce new native tissue. 31 Immune cells also play a key role in the signaling cascade leading to tissue regeneration, and appropriate engineering of the local immune response can boost the tissue regeneration. 32 For instance, monocytes and macrophages releasing cytokines including BMP-2, BP-4, and TGF-β1 support osteoblast differentiation and proliferation. 33 The current gold standard for understanding biocompatibility remains clinical trials, but essential information can also be derived from well-designed pre-clinical trials. When selecting the polymer, we have two conflicting interests; from a biological perspective, natural biopolymers are favorable due to higher biocompatibility, meanwhile synthetic polymers have more controllable properties, including swelling, degradation, phase transitions, and mechanical properties. 34 Additionally, synthetic polymers are more favorable from a regulatory and financial perspective. To balance these interests co-gel solutions such as PEG-HA 35

| Crosslinking/gelation
The next step is to form the gel-network by crosslinking the polymer chains. There are two options here, and the polymer can be physically or chemically crosslinked. Physical crosslinking is a reversible process where weak non-covalent interactions (e.g., van der Waals, hydrogen bonding, electrostatic interactions) keep the network stable. The advantage here is that the gel can be formed without using a crosslinking agent and the gel is easier to mold into the defect geometry. 39 Alternatively, chemical crosslinking (covalent bonds) can be used. The covalent bonds tend to convey to the gel's improved mechanical properties and higher stability. 40 The gel stability is a vital matrix design as long degradation times and the inability to be remodeled by the cells will hamper tissue growth. In contrast, a fast degradation time will leave an unfilled void after the gel degradation. 41 The degree of crosslinking, meaning the number of bonds that interconnect the polymers to each other, is an important parameter for the material properties. With a higher degree of crosslinking, we can expect a higher viscosity, stiffness, and longer degradation time. 42,43 It has early been established that with increased degree of crosslinking, the gel's ability to swell decreases. 44 The equilibrium degree of swelling affects a series of properties such as solute diffusion coefficient, mechanical properties, and the mobility of therapeutic agents. 45 From a translational perspective, chemical crosslinking means introducing new chemicals and at least one more chemical reaction. It must be proven that the biomaterial remains biocompatible and that there is not an increase of leachables such as unreacted crosslinker. Dialysis tends to be an efficient method for removing such F I G U R E 1 Schematic of the design process of hydrogels as medical devices for musculoskeletal application. The process consists of three design blocks, first, the hydrogel is developed, then any particles or other composite inclusions are added before the delivery strategy is chosen impurities, but the removal and biocompatibility must be proven. This is further discussed in Section 5.

| Composite design
Like with other biomaterial types, composite materials can be formed using hydrogels. This is a favorable strategy as the final material will have inherent properties from base materials in addition to the properties derived from the interaction between material components. In terms of hydrogels, this could be the introduction of fibers, for instance, to improve mechanical properties or guide tissue growth, or particles, for example, a ceramic phase which can boost bone regeneration. Li and coworkers 46 combined a hydrogel of thiolated HA and polyethylene glycol diacrylate (PEGDA) gel covalently crosslinked to fragmented, electrospun polycaprolactone fibers. The fibers gave improved mechanical properties compared to the HA-PEG gel alone, thereby they could mimic the mechanical properties of native fat tissue. Moreover, their in vivo trials with subcutaneous injection of the material in a rat and a rabbit model suggest improved macrophage polarization toward a pro-regenerative phenotype and enhanced angiogenesis.
The inclusion of an inorganic phase in the polymeric hydrogel material has been a popular strategy for bone regeneration. Chahal and colleagues 47 developed a PEG hydrogel with amorphous calcium phosphate particles. They demonstrated that the particles both gave a higher stiffness, and slowly released calcium and zinc ions into the solution, creating conducive properties for bone regeneration.
Although they observed a qualitative increase in gel mineralization, they could not demonstrate statistical significance. Furthermore, the human mesenchymal stem cells (MSCs) they used were unable to attach to the gel before they functionalized the PEG with RGD tripeptide motifs. This highlights the importance of choosing a polymer with high bioactivity to succeed clinically and demonstrates one of the shortfalls of most fully synthetic systems. Semi-synthetic systems, however, are promising as they allow for tunable properties such as gelation mechanism and adhesion to tissues. Researchers from the Langer lab 48 developed a cellulose hydrogel with PEG-block-poly (lactic acid) nanoparticles as non-covalent crosslinking nodes that gave the gel shear-thinning and self-healing properties. In vivo in mice (subcutaneously in the back) they demonstrated biocompatibility with a mild neutrophil-induced inflammation at day 3 and clearance by macrophages from day 7. A consistent release pattern was observed when particles were loaded with model dual-hydrophobic/hydrophilic drugs.
Wang and colleagues 49 formed a mechanically strong, transparent, and self-healing hydrogel by coating clay nanosheets with sodium polyacrylate and physically crosslinking it with dendritic G2 binder.
Any inclusion will make it, from a regulatory perspective, a completely new biomaterial. Therefore, it will require the standard omni-comprehensive testing due for any novel formulation. This includes the application of ISO 10993 family of standards that encompass biocompatibility testing up to clinical studies, and for biodegradable biomaterials the documentation requirements that degradation products do not accumulate in any body organs. 50

| Implantation method
Although the implantation method of the hydrogel is not directly a design variable affecting the gel properties, the hydrogel should be designed with implantation feasibility in mind. There are primarily two strategies of implantation in current use. The traditional is surgical incision implantation, where a surgeon cuts a flap through the patient's dermis and physically places the implant at the desired location. The advantage of this intervention is that the gel can be preshaped prior to the surgery and have higher mechanical stiffness. The disadvantage is that the incision surgery gives a longer hospitalization time, longer recovery time, increased postoperative pain, 51 and higher risk of bacterial infections. 52 Therefore, injectable solutions are attractive minimally invasive strategies that give less trauma, 53 less blood loss, shorter surgeries, and rapid recovery. 54 This brings its own technical challenges, as the gel must have low enough viscosity to be injectable through a needle or arthroscopic instruments. To have adequate viscosity during injection, it might be favorable to use a low degree of crosslinking, 43 a physically crosslinked gel exhibiting shearthinning properties, 48,55 or utilize in situ crosslinking of the hydrogel using methods such as click-chemistry, 56 ultrasound, 57 and photoinitiated crosslinking. 11,12 For in situ crosslinking, it is imperative to ensure that there are no adverse chemical reactions between the material and the surrounding biological tissue. For instance, thiol groups are naturally occurring in the body, so if a thiol-based Michael addition strategy is used for gelation, there is a risk of undesired cross-reactivity, oxidation, or metabolism. 40 This has inspired the focus on bioorthogonal chemistry, a class of high-yielding reactions based on selective transformation not commonly found in biology. 58 An innovative solution for injection of a hydrogel therapy is the Flowbone ® solution developed by researchers at EPFL in Switzerland.
They have developed a biphasic gel solution for bone regeneration where the first phase consists of covalently crosslinked HA with hydroxyapatite particles incorporated, that is carried in a second aqueous phase comprising more hydroxyapatite particles. 59 The biphasic system allows a low viscosity and thereby injectability. This solution also allows for the loading drugs such as bisphosphonates, 60 which is now under investigation in pre-clinical trials.
Other solutions chose a tactic where the crosslinking occurs in situ, such as Regentis Biomaterial's GelrinC ® , which is discussed below. The in situ strategy allows for a low viscosity during injection, while the high viscosity and mechanical properties are obtained after injection.

| APPLICATIONS
A series of hydrogel-based products have been approved for clinical use in the EU and the United States, particularly for viscosupplementation (VS) in joints for osteoarthritis (OA). Furthermore, regenerative gels are now emerging that in addition to providing temporary pain relief and functional improvement, attempt to regrow or support the regrowth of the tissue for a longer-lasting therapeutic effect. In this section, we describe some of the leading clinical products for VS. In addition, we will discuss the products that have undergone clinical trials or been commercialized in the EU or the United States to regenerate bone, cartilage, or NP tissue. The products we will discuss are summarized in Table 1. We present their application indications, therapeutic effect, delivery method, and composition. Apart from VS, the list is exhaustive to the authors' best knowledge but might suffer from lack of data availability as many manufacturers choose to keep data on file rather than publishing their results. With the introduction of the European EUDAMED database, this is expected to change within the EU market. Bone putties (DBM/inorganic particles in hydrogel carrier) have been excluded for bone regeneration products unless they are marketed as injectable gels.
Many manufacturers have chosen to not publish their findings but keep their data privately on file. This applies to the products AphaGRAFT ® , Kinex ® , AlloFuse ® , and Tactoset ® , meaning we have limited information on these products which can limit our discussion of these solutions.

| Cartilage treatment
An exciting area where injectable hydrogels have become an established treatment is cartilage degeneration in joints. This is pri- Patients with mild to moderate OA usually are treated with intra-articular injection of corticosteroids, as it provides an antiinflammatory effect. 93 However, corticosteroids are just capable of treating the symptoms, that is, reducing pain, but not able to stop the progress of OA. 94 Therefore, VS has become a popular treatment alternative as it provides a longer therapeutic effect. 95 For late-stage OA, arthroplasty is the preferred treatment, where the joint is partially or totally replaced with a prosthesis that is typically made of cobalt chrome or titanium alloys. 96 An alternative treatment is microfractures to release chondroprogenitor cells to the diseased location, but this tends to form fibrocartilage instead of desired hyaline cartilage. 97 The fibrocartilage has inferior mechanical properties than the native hyaline cartilage, 98  Recent clinical trials demonstrated that injection of HA has antiinflammatory and antioxidative properties, which can decrease the progression of OA. 106 This effect seems to be mediated through receptor signaling via binding with cluster determinant 44, toll-like receptors 2 and 4, intercellular adhesion molecule I, and layilin, providing a multifactorial mechanism. 107 Additionally, there are indications that high molecular weight HA promotes an anti-inflammatory response, meanwhile, low molecular weight HA favors an inflammatory response. 107 Altogether, intra-articular injections of HA-based VS have demonstrated an effect, and there is still room to tune the hydrogel composition to obtain solutions providing better lubrication with enhanced therapeutic benefit.
A recent commercialization is VS made from polyacrylamide such as Contura's Arthrosemid ® . Arthrosemid ® is a gel consisting of covalently crosslinked polyacrylamide, which is non-degradable. 66 It was used initially for veterinary application in horses with OA, 108 but recently the therapeutic effect has been demonstrated to be functional up to 52 weeks in humans. 67 As the material is non-degradable, the therapeutic effect is expected to be significantly longer. An in vivo subcutaneous rat model comparing the acrylamide gel to a HA gel as soft tissue fillers suggested significantly different in vivo behavior.
The acrylamide underwent cell infiltration by macrophages and fibroblasts and tissue integration, meanwhile, cell infiltration did not occur in the HA gel which was encapsulated by a thin fibrous layer. 109 The relevance of the model is limited as the study was conducted in a small animal with a subcutaneous application instead of intra-articular.

| Bone regeneration
Healthy bone is vital for structural stability in the musculoskeletal system, and defects result in pain, disability, and reduced mobility in individuals. Additionally, the treatment of bone defects is a tremendous burden to healthcare providers, estimating an annual cost of $5 billion in the United States alone. 118 Even though bone defects are rarely directly mortal, the trauma-induced can be hard to recover from. If we consider the case of hip fractures, for elder women (>65 years) there is a 10% likelihood of mortality within 3 months of a hip fracture. 119 Similarly, a larger meta-analysis demonstrated that the risk of mortality is increased by a 6-and 8-fold the first 3 months after hip fracture for older women and men (>50 years), respectively. 120 Nor are there any good treatment alternatives in these cases. In fact, another metaanalysis demonstrated that the mortality rate 1 year after hip fracture surgery is 24.5%, 121 suggesting two scenarios: (1) the current medical devices do not have an appropriate therapeutic effect for the elderly population, or (2) the current surgical procedure's invasiveness leads to a challenging recovery for elderly patients .
Bone defects can be widely different, and the products used depend on defect size and loading level. 122 Therefore, this section has been split into three subsections: (1) dental and maxillofacial,

| Dental and maxillofacial
Straumann's Emdogain ® dominates the dental market and has more than 20 years of clinical documentation. 124 Emdogain ® is based on a porcine enamel matrix derivative, a cocktail of proteins consisting of amelogenin (90%) and a few other nanomelogenin such as ameloblastin, enamelin, and tuftelin, carried in an aqueous gel solution composed of propylene glycol alginate. 39 Several of these proteins are identified as intrinsically disordered polypeptides with a one-to-many signaling effects in vivo and allow for the formation of multiple tissues in the injection location. 125 Emdogain ® has been proven to regenerate multiple periodontal tissues, including the osseo-like tissues, acellular cementum, 126 and alveolar bone, 127 in addition to connective tissues such as periodontal ligament. 128 The details of the therapeutic effect of Emdogain ® have been discussed in detail in our former review. 39

| Nucleus pulposus
Spinal fusion tends to be conducted due to DDD, where the IVD has degraded and lost its height or fractured. The IVD is to find between all the vertebra of the spine. It has three main components; the hydrogel-like NP in the core, surrounded by the annulus fibrosus (AF), and cartilaginous end plates (CEP) at the top and bottom ( Figure 4a). 146 The NP consists of approximately 50% (dry weight) proteoglycan proteins that play a vital role in binding water in the NP and shock absorbance. 147 During disc degradation, the concentration of proteoglycans decreases, causing a drop in stiffness. 146 This increases the risk of AF bulging, increases the compressive strain on the AF (Figure 4b), and increases the chances of peripheral failure of the end plates. 148 Therefore, a potential treatment of DDD would be to repair the NP.
A solution that has been approved for the European market is GelStix ® . GelStix ® uses a dehydrated polyacrylonitrile that is injected into the NP through a 22-G needle in the form of a filament, where it gets hydrated from the surrounding body liquids and expands 10-fold. 84 In a 12-month follow-up with 29 patients, 86.2% rated the procedure as very good or good, and pain relief was observed already after 1 month. 84 However, there have been reported complications associated with this procedure. Durdag and colleagues reoperated a woman with a GelStix ® implanted as she was admitted with severe radicular pain. 149 The pain was linked to a fragment of implant that had penetrated through an annual tear and caused compression to the spinal root. The authors speculate that the implant may have been initially wrongly placed in the AF, highlighting the importance of the correct placement of the implant.
HA with a similar composition to the solutions used for VS has been used for treatment of the NP. In a 24-week follow-up period, Mazza and co-workers observed relief from chronic lower back pain due to DDD compared to the baseline. 85 They had two patients drop out due to adverse events, but this is not believed to be related to the treatment. However, the clinical efficacy is proven only over a short time period. Considering the surgical risk related to bypassing vital organs during injection, this therapy can come short when evaluating it using a cost-benefit analysis. Hence a longer-lasting therapy should be investigated.
Two other solutions have been tried clinically, but seem to have been discontinued. The NuCore ® gel for NP replacement consists of elastin and silk co-polymers that are crosslinked in situ. 89  Since neither CryoLife nor Spine Wave has disclosed why their products were discontinued, it is not feasible to conclude why they failed to perform in the clinic. devices, demonstrating that the device is safe and efficient, which is significantly cheaper than introducing a new device. In the case of class III, the 510(k) allows the manufacturer to partially bypass the premarket approval application, meaning they do not need to run a clinical investigation, but this is not applicable for the VS products or dental biological materials discussed here. When the 510(k) is not applicable for the class III devices, the product needs to be evaluated on a case-by-case basis by the authorities (US-FDA).

| REGULATORY CLASSIFICATION AND CONSEQUENCES
In the review, we have focused on discussing hydrogels as medical devices. However, they can also be classified as medicinal products if their main mechanism of action is through pharmacological, metabolic, or immunological means 6 ; this would lead them to the so-called "drug approval process." A couple of hydrogels that are used for the above-described musculoskeletal treatments are classified by the European Medical Agency and the FDA as medicinal products (biologics/drugs) instead of medical devices as they have the characteristics of combinatory products, Advanced Therapy Medicinal Products (ATMP). A summary of these can be found in Table 2.
Over the last couple of decades, there has been a drastic change in the design rationale of orthopedic biomaterials. From passive structures designed for minimal interaction with the surrounding tissue, for example, titanium-based hip implants, the current generation of biomaterials is designed to actively interact with the surrounding tissue, such as scaffolds for tissue regeneration that stimulates tissue growth.
This means that the product's mechanism of action starts approaching that of medicinal products, which will change the applicable regulation framework. 154 Hence engineers need to carefully consider regulatory classification when designing hydrogels. If a hydrogel solution is classified as a medicinal product, it increases the documentation and overall market entry requirement and requires larger and more costly clinical trials. Compared to medical devices, the therapy will take significantly longer time for clinical translation, the R&D investment costs will increase drastically, and the product will eventually be sold at a higher price to the healthcare providers. Moreover, there will be longer product cycles, which means less innovation. In the United States, it takes on average 12 years from pre-clinical trials to market approval for drugs while it only takes 3-7 years for medical devices, and the development costs will increase from the range of tens of millions of dollars for medical devices up to the excess of $1 billion for pure drugs. 155,156 Products where a medical device (i.e., the gel) carries a therapeutic agent such as growth factor or expanded cells no longer gets its  Notably, the new EU MDR requires a post-market surveillance register for medical devices (EUDAMED). This is inspired by the successful implementation of orthopedic device registries and the quality of the data these have provided. 4 With this registry, the regulation requires continuous data gathering and analysis. More specifically the MDR article 83 states 162 : "The post-market surveillance system shall be suited to actively and systematically gathering, recording and analysing relevant data on the quality, performance and safety of a device throughout its entire lifetime, and to drawing the necessary conclusions and to determining, implementing and monitoring any preventive and corrective actions." The medical device industry is characterized by a lot of small, niche suppliers. In Europe, out of 33,000 medical technology companies, 95% are small or medium enterprises (<250 employees), and a majority are small or micro-sized companies (<50 employees). 164 The limited manpower makes it challenging for these companies to designate and dedicate personnel for the post-market clinical follow-up.
This provides a golden opportunity for academic researchers to collaborate with these companies to analyze the clinical data, and academics can use their understanding of fundamental biological and clinical mechanisms to explain the collected observations, for example, evaluating porcine versus bovine gelatin in the bone graft SmartBone. 165 If the data are published, it will indeed help the wider research community. Meanwhile, the companies will benefit from this as they can leverage experienced personnel to analyze and explain complex data.

| CONCLUDING REMARK AND FUTURE DIRECTION
There is a tremendous discrepancy between the intensity of academic research on hydrogels and the number of products that have been clinically translated for the treatment of musculoskeletal defects.
When developing hydrogels, it is crucial to consider the clinical potential of the material, and here pre-clinical and clinical trials are key in predicting whether a material candidate will make it past the evalua- In vitro testing is very important for understanding isolated mechanisms. However, in our experience [167][168][169] there are major differences in response to biomaterials during in vitro tests, where single cell types are used, and in vivo, where there is an assortment of cell types interacting. 33,170 Although there is progress in technology such as organ-on-chip 171,172 or co-cultures, 173 they are yet not capable of mimicking the complexity of tissue response to biomaterials. Simultaneously, animal trials should be kept to a minimum for ethical and economic reasons. To obtain adequate documentation and keep animal trials to a minimum, care should be taken in acquiring high quality in vivo data. The ISO 10993-6 (Test for local effect after implantation) requires only local microscopic assessment using histology. Using only this method gives an incomplete picture as conventional histology does not give spatial information or confirm certain biomarkers. 174 Hence, utilizing additional methods such as cone beam computed tomography, 167 microCT (μCT), 167 immunohistochemistry, 167,[175][176][177] small-angle X-ray scattering , 167,178 X-Ray diffraction analysis , 167,178 and more newly developed techniques such as fluorescent labeling of abundant reactive entities, 167 optical photothermal infrared microscopy, 167 and nanoscale atomic force microscopy-infrared 167 can give a comprehensive understanding of the material's mechanism of action. Furthermore, there has been an increased focus on the use of intravital microscopy such as fluorescence lifetime imaging microscopy and Raman spectroscopy as their subcellular resolution (approx. 500 nm) allows for studying in detail in vivo host response to implants and for monitoring of implant biology over time in small animal models. 30