Biomaterials for Orthopaedic Diagnostics and Theranostics

Abstract

Despite widespread use of conventional diagnostic methods in orthopaedic applications, limitations still exist in detection and diagnosing many pathologies especially at early stages when intervention is most critical. The use of biomaterials to develop diagnostics and theranostics, including nanoparticles and scaffolds for systemic or local applications, has significant promise to address these shortcomings and enable successful clinical translation. These developments in both modular and holistic design of diagnostic and theranostic biomaterials may improve patient treatments for myriad orthopaedic applications ranging from cancer to fractures to infection.

Clinical Rationale for Orthopaedic Diagnostics and Theranostics

Orthopaedic diseases and disorders are the second leading cause of disability worldwide and result in $880 billion in direct healthcare costs annually in the US alone [1,2]. As life expectancy increases, bone-related diseases and disorders have become increasingly prevalent. This trend will have significant impact on the rates of conditions such as osteoporosis, which affects >50% of Americans over the age of 50 [3].

Early and efficient detection and treatment of bone-related diseases and disorders is important to limit morbidity and mortality. For example, metastasis of aggressive cancers, such as osteosarcoma, severely impacts survival rates. Indeed, osteosarcoma survival rates fall from 70–80% to only 20–30% after metastatic progression [4]. Early diagnosis is critical for improving overall prognosis, as time to osteosarcoma diagnosis is correlated with greater rates of metastases and poorer prognosis [5,6]. Furthermore, early initiation of treatment can delay onset, number, and size of pulmonary metastases, increase the success of tumor resections, and improve overall survival [4,7,8]. Additionally, bone metastases from lung, prostate, and breast cancer require early detection to circumvent severe complications such as pathologic fractures or spinal cord compression [9].

Orthopaedic imaging plays a key role in current clinical diagnoses and disease progression and/or treatment monitoring. Modalities such as radiographs (x-rays), computed tomography (CT), magnetic resonance imaging (MRI), ultrasound, and nuclear imaging studies, including scintigraphy, single-photon emission CT (SPECT), and positron emission tomography (PET), are the mainstays of bone-related diagnostics. These techniques have distinct advantages and limitations, as detailed in Table 1. Specific to orthopaedics, features of bone-related disorders can be nonspecific and overlap with other etiologies, making accurate diagnosis a challenge [10]. Surgical management of bone-related pathologies can lead to anatomic changes and often requires hardware, which can also significantly interfere with image quality, limiting treatment monitoring [10]. Moreover, traditional diagnostic techniques may have a limited ability to detect bone disorders at early stages when intervention is most efficacious at preventing or mitigating progression [10,11].

Table 1:

Advantages and Disadvantages of Clinical Diagnostic Modalities^*

Modality	Advantages	Disadvantages
Radiography	High spatial resolution	Uses ionizing radiation (minor exposure)
(x-ray)	Portability	Superimposition of structures
	Less expensive	Lower sensitivity
CT	Volumetric data with multiplanar/3D reconstructions	Uses ionizing radiation (moderate exposure)
	High spatial resolution	Lower soft tissue contrast compared to MRI
	Gives detailed information about complex fractures	Less useful for soft tissue evaluation
	Few contraindications when compared to MRI
MRI	High soft tissue contrast/spatial resolution	Expensive
	Does not use ionizing radiation	Longer scanning time
	Better for evaluation of occult fractures, infections, articular cartilage, ligaments, and soft tissues	Image artifact from metal surgical implants
		Contraindications (pacemakers, claustrophobia, etc.)
Ultrasound	Does not use ionizing radiation	Operator dependent
	Less expensive	Limited evaluation of deep structures
	Portability	Lower resolution
	Ability for dynamic examinations and procedural guidance
Nuclear Medicine (Scintigraphy, SPECT, PET)	Accessibility to functional information	Uses ionizing radiation/radiotracers
	High sensitivity	Low specificity
		Low spatial resolution/anatomic localization
		Expensive

^*Adapted from Kamar and Hayashi (2016) [12]

CT=Computed tomography; MRI=Magnetic resonance imaging; SPECT=Single-photon emission computed tomography; PET=Positron emission tomography

Given the limitations of traditional bone diagnostic techniques, there is clear clinical rationale for developing advanced diagnostics and theranostics. In particular, biomaterials designed to target or localize to bone or disease sites are promising to increase the precision and prognostic value for cancer, osteoporosis, non-unions, fracture, and/or infection (Figure 1A). Furthermore, orthopaedic theranostics may have even greater clinical impact. Multimodal imaging can be employed to overcome the limitations of individual modalities, mediated through the use of multifunctional biomaterials. Herein, we discuss current advances and the promise in future biomaterials designs that can be used systemically or locally for diagnosing and treating for bone-related disorders (Figure 1B, C).

Figure 1: Overview of clinical needs for diagnostic/theranostic biomaterials and example materials.

A) Representative clinical images of orthopaedic applications. B, C) Systemic and local biomaterials highlighted in this review. PLGA: Poly(lactic acid-co-glycolic acid), PCL/PS: Poly(caprolactone)/calcium silicate. Porphysome is reproduced from [13] (https://pubs.acs.org/doi/10.1021/nn400669r) with permission. Further permission related to the material excerpted should be directed to the ACS. BP-targeted PLGA Nanoparticles reprinted with permission from [14]: Low SA, Galliford CV, Yang J, Low PS, Kopeček J. Biodistribution of Fracture-Targeted GSK3β Inhibitor-Loaded Micelles for Improved Fracture Healing. Biomacromolecules. 2015;16(10):3145–53. doi: 10.1021/acs.biomac.5b00777. Copyright 2015 American Chemical Society. Alkaline phosphatase detecting PCL/PS Scaffold Originally published in [15] reprinted from Chemical Engineering Journal, 408, Yang C, Gao X, Younis MR, Blum NT, Lei S, Zhang D, Luo Y, Huang P, J Lin J, Non-invasive monitoring of in vivo bone regeneration based on alkaline phosphatase-responsive scaffolds, 127959, Copyright 2021, with permission from Elsevier under CC BY-NC 4.0 license #5036610232181. Oxygen-detecting poly(caprolactone) scaffolds reprinted with permission from [16]: Schilling K, El Khatib M, Plunkett S, Xue J, Xia Y, Vinogradov SA, Brown E, Zhang X. Electrospun Fiber Mesh for High-Resolution Measurements of Oxygen Tension in Cranial Bone Defect Repair. ACS Appl Mater Interfaces. 2019 Sep 18;11(37):33548–33558. Copyright 2019 American Chemical Society. Strontium-doped hydroxyapatite-collagen scaffold image [17] is reproduced under CC BY-NC 4.0 license #5036610940442. Center skeleton is from [18].

Biomaterials design considerations for bone diagnostics and theranostics

Biomaterials, including polymers [19,20], liposomes [13,21], gold nanoparticles (NPs) [22–24], mesoporous silica (MeSi) NPs [25–27], quantum dots (QDs) [28,29], upconversion (UC) NPs [30–32], hydroxyapatite [33–39], and superparamagnetic or ferromagnetic elements: nickel, iron (SPIONs), cobalt, and their combinations [40–42], are commonly used for diagnostics and theranostics (Figure 2, Table 2). Indeed, a recent review discusses diagnostic and theranostic biomaterials development for bone tumors [43]. However, far fewer approaches exist for other orthopaedic applications, which have unique diagnostic and theranostic requirements including deep and dense tissue penetration, robust, tissue-specific accumulation, and confounding factors from implants or other treatments. Diagnosing osteoporosis, for example, requires methods that can accurately measure skeletal biomechanics and physiochemical properties (i.e., size, shape, structural properties), while diagnosing osteomyelitis may be challenged by orthopaedic hardware and anatomical location [44]. These requirements are met through multifunctional biomaterials designed for both systemic administration and local applications that primarily enhance and extend the capabilities of existing orthopaedic diagnostic techniques.

Figure 2: Schematic representation of systemically delivered NP-based biomaterials used in diagnostics and theranostic for orthopaedic applications detailing the features that can be modulated.

SWCNT, single-walled carbon nanotubes; QDs, quantum dots; UCNPs, upconversion nanoparticles; GNPs, gold nanoparticles, SPIONs, superparamagnetic nanoparticles, MeSiNPs, mesoporous silica nanoparticles; HA NPs, hydroxyapatite nanoparticles. Figure adapted from [49].

Table 2:

Advantages and Limitations of Existing and Emerging Biomaterials Diagnostics and Theranostics [50–57]

Biomaterial	Advantages	Limitations
Polymers	Flexible compositions, functionalities, and morphologies to enable drug/tracer incorporation and targeting	Translation can be challenging due to difficult scale-up of manufacturing processes
	Tunable size, shape, responsive behavior, and degradation	No inherent diagnostic capabilities
Lipid-based NPs	Flexible compositions, functionalities, and morphologies to enable drug/tracer incorporation and targeting	No inherent diagnostic capabilities
Gold NPs	Favorable photostability	Limited surface area for drug/tracer, targeting group, and stabilizing molecule incorporation
	Tunable absorptive/scattering properties based on size and shape	Rapid clearance
	Excellent biocompatibility	Toxicity
	Simple conjugation of targeting groups and/or therapeutic molecules via thiol chemistry
Superparamagnetic iron oxide (SPIONs)	Biocompatible	Dose accumulation over time leading to toxicity
	Exhibit in vivo biodistribution tunability based on size and surface modifications
	Detectable via MRI, which enables robust resolution
	Induction of localized heating in magnetic fields
Upconversion nanoparticles (UCNPs)	Absorb multiple photons to produce high energy anti-Stokes luminescence	Non-negligible heating of exposed tissues
	Minimal tissue autofluorescence	Low brightness relative to other NIR fluorescent probes
	Enable multiplexed imaging
	Highly resistant to photobleaching
Quantum dots	High quantum yield	Sometimes formed from cytotoxic elements
	Photostable	Rapid clearance
	High signal-to-noise ratio
	Simultaneous excitation of multiple wavelengths
Hydroxyapatite	Biocompatible	Rapid clearance
	Bioactive
	Osteoconductive
	Excellent drug loading capabilities
Mesoporous silica	High cargo loading capacity, controllable release	Cargo leakage
NPs	Excellent chemical, thermal, and mechanical stability	Biocompatibility and toxicity
	Tunable size and porosity
	Flexible morphology

Systemically administered biomaterials for bone diagnostics and theranostics

Systemically administered biomaterials, often in the form of NPs, are commonly employed for bone diagnostics/theranostics (Figure 2 and Table 2). NPs have many advantages as diagnostic/theranostic biomaterials platforms. NPs can be systemically administered noninvasively and, if properly designed, can achieve tissue specificity through targeting moieties [43]. NPs are inherently multifunctional and/or modular to enable tissue targeting/selectivity, enable sustained and/or responsive drug release, and protect sensitive drug payloads from degradation [45]. NP chemistry can also be altered to modulate circulation half-life, for example, through poly(ethylene glycol) functionalization [46,47]. NPs can be developed to deliver synergistic, multidrug combinations of myriad drug cargos (hydrophobic or hydrophilic drugs/contrast agents, nucleic acids, etc.), and/or to localize heating or radiation in conjunction with multimodal imaging [45]. Additionally, some NP compositions, namely metallic NPs, are inherently magnetic or fluorescent for imaging [48].

Facile incorporation of bone targeting, either through passive or active mechanisms, is another advantage of NPs that can lead to the specificity and contrast necessitated by bone diagnostics/theranostics (Figure 2). Indeed, systemically delivered small molecule biodistribution to bone is poor with <1% of doses successfully reaching bone [14]. Tissue specificity arises from the incorporation of targeting moieties and/or enhanced permeability and retention (EPR) due to tumor growth, infection, or injury [58–61]. For example, first generation small molecule radiotracer-based bone diagnostics were developed based on molecules with bone affinity including ¹⁸F-NaF, ^99mTc-methylenediphosphonate (MDP), and ^99mTc-hydroxymethylenediphosphonate [62–66]. Similarly, facile incorporation of bone targeting moieties is a key asset of NPs to provide specificity and contrast necessitated by bone diagnostics/theranostics (Figure 2). For bone targeting of NPs, bisphosphonates (BP) are commonly used. BP bind generally to bone tissue due to high affinity for hydroxyapatite [67,68] and also to regions of high metabolic activity, such as primary bone tumors (e.g., osteosarcoma) or metastases [69,70]. Other targeting groups have been explored, including phytic acid, aspartic acid and glutamic acid and (co)polypeptides thereof, tetracycline, as well as aptamers and other peptides with affinity to the bone matrix or relevant cells, as recently reviewed [43].

Macroscale biomaterials for diagnostics and theranostics

Macroscale or bulk biomaterials placed locally within or juxtaposed to bone during surgical procedures also have significant value for diagnostics and theranostics. These include tissue engineering scaffolds, bone fixation devices or implants, and drug reservoirs using a variety of biomaterials including poly(caprolactone), poly(lactic acid), poly(glycolic acid), poly(methyl methacrylate), poly(butyl terephthalate), poly(carbonate), hydroxyapatite, calcium phosphate, and calcium silicate [71,72]. The general design principals and biomaterials used for implantable orthopaedic devices can be found in recent reviews [71–73]. Common orthopaedic biomaterials are adapted for diagnostic/theranostic applications through the incorporation of contrast agents and responsive moieties, which enables sensing of the local microenvironment, therapeutic efficacy, or the rate and/or extent of degradation of scaffolds. Due to localized placement, macroscale biomaterials are subject to different delivery constraints (e.g., surgical placement or injectable localization) compared to systemically administered biomaterials, which can lessen the risk for off-target effects and background and may also enable use of more varied materials with greater quantities/varieties of detection moieties. Diagnostic capabilities are often employed in biomaterials development in the pre-clinical stage to better understand in vivo interactions and provide examples of potential future clinical applications.

Biomaterial Diagnostics and Theranostics in Development for Orthopaedic Applications

The goal of biomaterials-based diagnostics is to improve existing detection modalities, either through enhanced contrast or increased pathological specificity. Examples of diagnostic and theranostic biomaterials developed for both systemic and local orthopaedic applications since the year 2010 are highlighted in Table 3, with selected examples discussed in subsequent sections.

Table 3.

Examples of biomaterials explored for bone diagnostics and theranostics from 2010–2021. Additional examples pertaining to bone tumor diagnostics and diagnostics can be found in the following review [43].

Biomaterial Type	Diagnostic/Therapeutic Entity	Surface modification	Rationale for Use in Bone Applications	Clinical application	Diagnostic Modality*	Ref
SPIONs	Iron-oxide NPs	1,5-dihydroxy-1,5,5-tris-phosphono-pentyl-phosphonic acid (di-HMBPs)	High affinity for calcium ions/hydroxyapatite	Osteoporosis	MRI	[74]
	Iron-oxide NPs	Alendronate	High affinity for hydroxyapatite	Bone metabolic activity	MRI	[75]
	99mTc labeled iron-oxide NPs	Alendronate	High affinity for hydroxyapatite	Bone	SPECT/PET-MRI	[76,77]
	99mTc labeled PEGylated iron-oxide NPs	Bisphosphonate	High affinity for hydroxyapatite	Bone	SPECT/PET-MRI	[77]
	Iron-doped hydroxyapatite NPs	N/A	Bone substitute	Bone	SPECT/PET MRI	[34]
Carbon nanotubes	99mTc labeled carbon nanotubes	Alendronate	High affinity for hydroxyapatite	Active bone metabolism	Photoacoustic imaging	[78]
Quantum dots	Quantum dots	Various antibodies	Binding to unique cell populations in bone marrow	Targeted cell imaging	NIR	[79]
	Ag2S QD/ Doxorubicin	Alendronate	High affinity for hydroxyapatite	Bone tumors	NIR	[80]
Gold NPs	Gold NPs	Glutamic acid	Targets microcracks by chelating with calcium ions	Damaged bone	CT	[81]
	Gelatin methacrylate -gold NP scaffold	N/A	N/A	Bone defects	CT	[20]
	Gold nanorods	Tumor specific oligopeptides	Binding to osteosarcoma cells	Osteosarcoma	Photoacoustic imaging	[82]
Mesoporous silica NPs	Mesoporous silica NPs loaded with ammonia borate	N/A	Nanocomposites respond to acidic environment of the tumor to release H2 improving contrast between osteosarcoma and surrounding healthy bone	Osteosarcoma	CT	[33]
	Mesoporous silica NPs/ gold nanorods	Zoledronic acid	High affinity for hydroxyapatite	Bone metastasis	Photoacoustic imaging	[83]
	Mesoporous silica NPs containing an iron oxide core-immobilized with BMP-2, coated with calcium phosphate/ Bone morphogenic protein 2 (BMP-2)	N/A	Resemblance to native bone	Critical sized defects	MRI	[84]
	Mesoporous silica-coated bismuth sulfide NPs/ Doxorubicin	RGD-peptide	Target tumor vasculature and tumor cells	Osteosarcoma	NIR/CT	[85]
Hydroxyapatite	Silicate-substituted HAp doped with Eu(III) and Bi(III)A	N/A	Bone substitute	Bone	Luminescence	[39]
	Calcium deficient hydroxyapatite scaffold labeled with NIR probe	N/A	Bone substitute	Monitor bone healing	NIR	[86]
	Hydroxyapatite NPs/ Ho-166	N/A	High affinity to bone	Bone cancer	Unclear	[38]
	Folic acid modified hydroxyapatite NPs/ 64Cu	Medronic acid (MDP)	High affinity to areas of active bone metabolism	Osteosarcomas and other bone disorders	PET	[36]
	Nano-hydroxyapatite rods/ Molybdenum oxide	N/A	Bone substitute	Bone infection	Fluorescence	[37]
	Eu(III)/Gd(III) doped hydroxyapatite nanorods/ Ibuprofen (as model drug)	N/A	Bone substitute	Bone	Luminescence/MRI/CT	[35]
	Iron-doped hydroxyapatite alginate-gelatin scaffold/ Bone morphogenic protein 2 (BMP- 2)	N/A	Osteoconductive properties of scaffold	Localized monitoring of cell infiltration in bone	MRI	[87]
Upconversion nanoparticles (UCNPs)	18F labeled NaGdF4:Yb, Er UCNPs	Etidronic acid, alendronic acid, and nitrile (trimethylphosphonic acid)	High affinity for hydroxyapatite	Bone	MRI/PET	[88]
	Yb(III)/Ho(III) doped UCNPs	N/A	Apatite component of UCNP mimics bone	Bone regeneration	Luminescence	[30]
	NaYF4:Yb3+,Er3+ UCNPs	Positive and negative charged polymers	Use for tracking without affecting intrinsic cell properties	Cell localization	Fluorescence	[31]
	NaYbF4:Gd3+/Er3+ UCNPs	Iminodiacetate	Chelates exposed calcium ions	Damaged bone	Gemstone spectral CT	[89]
	Mesoporous silica-coated upconversion NPs doped with gadolinium (III)/ Plumbagin and poly (acrylic acid)	Zoledronic acid	High affinity for hydroxyapatite	Bone metastasis	MRI/luminescence	[32]
Polymer-based systems	Electrospun polycaprolactone fibers encapsulating PtP-C343 (phosphorescent probe)	N/A	Electrospun fibers mimic ECM	Bone healing	Two-photon phosphorescence lifetime microscopy	[16]
	PLGA-PEG loaded with iron-oxide NPs and NIR dye	Alendronate	High affinity for hydroxyapatite	Bone	MRI/NIR	[90]
	Fluorescein isothiocyanate labeled PEGylated Poly(y-benzyl-l-glutamate)(PBLG)	Alendronate	High affinity for hydroxyapatite	Bone	Fluorescence	[91]
	Poly(trimethylene carbonate)-b-poly(glutamic acid) loaded with SPIONS	HER2 antibody	Tumor targeting	Bone metastasis	MRI	[19]
	99mTc labeled polymers	N/A	Binds to bone surface	Bone metastasis	PET	[92]
	SPION coated with chitosan-PEG copolymer	HER2 antibody	Binds to metastatic tumor cells	Bone metastasis	MRI	[19]
	Polycaprolactone/calcium silicate, (PCL/CS) scaffold labeled with hemicyanine dye	N/A	Alkaline phosphatase (ALP) responsive dye	Monitor bone healing	NIR/photoacoustic imaging	[15]
	Poly(lactide-co-glycolide) (PLGA) loaded with SPIONS/ Paclitaxel	Alendronate	High affinity for hydroxyapatite	Bone tumors	MRI	[93]
	HAp composite PLGA scaffold/ SPIONs	N/A	Bone substitute	Bone defect	X-ray	[94]
	Iron-doped polydopamine NPs/ 7-ethyl-10- hydroxycamptothecin (SN38)	Alendronate	High affinity for hydroxyapatite	Bone tumor/osteolysis	MRI	[95]
	Chitosan-grafted PEG copolymer with SPION core	HER2/neu	Tumor targeting	Bone metastasis	MRI	[96]
Lipid-based systems	64Cu-porphysomes	N/A	Binds to metastatic tumor cells	Bone metastasis	PET/fluorescence	[13]
	PEGylated liposomes loaded with CdSe QDs and iron oxide NPs	cRGDyk peptide	Binds to metastatic tumor cells	Bone metastasis	MRI/fluorescence	[21]

Abbreviations: MRI = magnetic resonance imaging; SPIONs = superparamagnetic iron oxide nanoparticles; HAp = hydroxyapatite; NIR = near infrared imaging; PET = positron emission tomography; ECM = extracellular matrix; HER2 = human epidermal growth factor receptor 2; PLGA = poly(lactic acid-co-glycolic acid); PEG = poly(ethylene glycol); Yb = Ytterbium; Ho = Holmium; Gd = Gadolinium; Er = Erbium; Eu = Europium; Bi = Bismuth; CT = computed tomography; RGD = arginine-glycine-aspartic acid.

^*denotes imaging modality used in the paper but is not an exhaustive list of the potential diagnostic applications.

Systemic Diagnostics and Theranostics

Several examples of orthopaedic diagnostics and theranostics in development involve systemic delivery of biomaterials (Table 3). Systemic diagnostic and theranostics are defined here as technologies injected intravenously, subcutaneously, intradermally, or otherwise. One critical hurdle for systemically delivered diagnostics/theranostics is tissue specificity to ensure high levels of contrast between dysfunctional and healthy tissues for reliable disease detection. Despite this hurdle, a variety of designs developed in the absence of targeting ligands have resulted in dramatic improvements in traditional diagnostics. For example, porphyrin-lipid conjugates combined with small fractions of radioactive ⁶⁴Cu were used to detect metastatic bone lesions via porphyrin fluorescence and ⁶⁴Cu PET/CT imaging in a prostate cancer animal model [13]. While traditional PET scans are limited to detection of metastatic lesions larger than 1 cm, the porphysome technology improved sensitivity to 2 mm. Porphysomes also accumulated at naïve bone, suggesting inherent bone diagnostic capabilities, which may be useful for adapting the technology to other orthopaedic applications (e.g., bone mineral density, stress fracture detection). In another example, PEGylated MeSiNPs were used to deliver ammonia borate (AB), which releases H₂ in acidic environments, resulting in negative CT contrast [33]. While a clinical contrast agent was unable to differentiate between osteosarcoma and healthy bone in a rat model, intravenous injection of MeSiNPs enabled 20x greater contrast on CT [33], therefore highlighting the utility of MeSiNPs-AB diagnostics.

To achieve the goal of high tissue specificity and diagnostic or theranostic sensitivity, bone-targeted biomaterials have been developed based on existing clinical diagnostic modalities. For example, gold NPs functionalized with bone-targeting glutamic acid were developed to non-invasively detect bone microdamage, which is indicative of impending bone fracture but is undetectable using x-ray [81]. Ex vivo x-ray revealed preferential binding to damaged bone owing to glutamic acid binding to exposed calcium versus healthy bone tissue, suggesting the utility of gold NPs for bone diagnostics and theranostics. For cell targeting, HER2 antibody conjugated PEGylated polymersomes loaded with SPIONs were used to detect tumor boundaries in bone metastasis compared to untargeted controls in a breast cancer metastasis animal model [19]. Following the idea of tissue targeting, theranostic alendronate-functionalized iron-doped poly(dopamine) NPs were used to image tumor shrinkage via MRI [95]. Compared to untargeted controls, the NPs suppressed tumor growth and reduced osteolytic bone damage in an orthotopic bone tumor model due to combined chemo-photothermal therapy of iron and the therapeutic efficacy of the loaded drug, 7-ethyl-10-hydroxycamptothecin [95]. BP-functionalized, PEGylated ^99mTc-SPIONs exhibited enhanced accumulation at bone compared to untargeted controls [77] and leveraged multimodality to overcome SPION sensitivity issues, enabling successful longitudinal monitoring in animal studies. In the absence of the radiolabel, improved BP-PEG-SPION sensitivity was attributed to the small NP size (~20 nm), hydrophilicity of BP and PEG that allow for proton relaxation at the iron oxide NP surface, and the presence of the PEG coating, which prevented aggregation. This approach may be useful to enhance diagnostic capabilities of MRI for detecting osteoporotic bone and bone metabolic activity.

Emerging technologies such as photoacoustic (PA) and NIR imaging combined with biomaterial diagnostics and theranostics present unique opportunities for orthopaedic applications. PA imaging can reach tissue depths up to 5–6 cm with outstanding spatial resolution of ~5 μm. Improved capabilities of PA was exploited using PEGylated gold nanorods targeted with osteosarcoma-specific peptides to visualize neovascularization with high contrast via PA imaging, enabling differentiation between tumor and healthy tissue [97]. To diagnose and treat bone metastasis in an animal model of breast cancer, gold nanorods were incorporated within MeSiNPs functionalized with zoledronic acid and imaged photoacoustically [83]. MicroCT revealed tumor size, and osteolysis was reduced compared to untreated controls. NIR imaging allows for deep tissue penetration with minimal tissue autofluorescence. To diagnose and treat bone metastases, zoledronic acid targeted MeSi-coated UCNPs doped with gadolinium were injected in a bone metastatic breast cancer model [32]. Upconversion luminescence and NIR imaging revealed that loaded UCNPs reduced tumor size compared to empty UCNP controls. The theranostic effects of UCNPs were further verified using microCT, which revealed reduced osteolysis [32]. Similarly, QDs have been incorporated within cell-targeted NPs to characterize the heterogeneous bone marrow cellular repertoire [79]. To achieve dual-modality, RGD-functionalized liposomes co-loaded with iron-oxide and CdSe QDs were developed [21]. In a prostate cancer bone metastasis model, QD MRI signal was 1.5-fold higher than iron-oxide NPs alone and tumor fluorescence was higher than untargeted liposomes. QDs have also been used to detect prostate cancer-related delta/notch-like epidermal growth factor-related receptor expression, which has prognostic value for bone metastases [98]. Core-shell silica NPs (C dots) are emerging as alternative materials to quantum dots [99]. This class of biomaterials covalently integrate organic fluorophores into the core of core-shell silica NPs resulting in enhanced brightness (~2–3x vs. QDs) and photostability. For example, C dots were recently used to label cancer cells to study early-stage bone metastasis [100].

Local Diagnostics and Theranostics

Implantable biomaterials are adapted to local diagnostics or theranostics through the addition of therapeutics or contrast agents (Figure 1). There are several recent examples of bone diagnostics incorporated within traditional tissue engineering scaffolds. Electrospun poly(caprolactone) scaffolds encapsulating porphyrin-based sensors for oxygen tension, which is correlated with healing, have been used to monitor bone regeneration using two-photon microscopy [16]. A limitation of this approach is that sensitivity may decrease in humans in which scaffold placement will be deeper versus murine models. In an alternative system, MRI was used to image gadolinium-doped HA NPs incorporated into electrospun poly(caprolactone) scaffolds to track in vitro bone regeneration [101] as well as nano-hydroxyapatite-Alginate-Gelatin scaffolds incorporating SPIONs for detection of cellular infiltration and scaffold mineralization [87]. Bone morphogenic protein 2 was immobilized within iron oxide core mesoporous silica beads and incorporated within a calcium phosphate cement, enhancing contrast and improving bone regeneration for up to 8 weeks following theranostic implantation [84]. Finally, gold NPs entrapped within gelatin methacrylate (GelMA) hydrogels were developed to enable imaging during the process of bone regeneration within a condyle defect [20], enabling greater resolution of bone microarchitecture compared to GelMA hydrogels alone. The ability to assess bone regeneration and/or integration using engineered scaffolds can improve monitoring and enable earlier detection of treatment failures.

Future Directions

Biomaterials-based diagnostics and theranostics have great promise for orthopaedic applications. However, significant opportunities and challenges remain. Refinement of biomaterials designed for diagnostics and theranostics is necessary to meet the demands of orthopaedic applications. In general, conventional theranostic and diagnostic biomaterials are combinations of existing technologies. This modular approach that integrates therapeutic, diagnostic, and targeting moieties enables a high degree of tunability in the resulting diagnostic/theranostic at the cost of complexity. This principle also applies to diagnostic agents embedded within biomaterials for local applications, which allows independent tuning of the scaffold properties and detection modalities. Complementary to this trend of isolating functionalities is that of more holistic design tailored to orthopaedic diagnostics/theranostics. As opposed to adapting existing formulations for additional diagnostic/theranostic functionality, inherently multifunctional biomaterials allow highly integrated designs with fewer components that may ultimately allow for greater reproducibility in design, manufacturing, and testing. An example of holistic design is ⁶⁴Cu-porphysome technology, where the biomaterial complexes with Cu and is also fluorescent. Not only will holistic design likely provide greater efficacy but may also streamline FDA approval due to its simplicity.

Several difficult to detect orthopaedic conditions are well suited for diagnosis and treatment with high-contrast and high-specificity NPs, including tumors, osteomyelitis, and osteoporosis. Nevertheless, myriad challenges remain for systemically administered biomaterials including NPs. For example, bone possesses unique barriers to NP delivery including limited vascularization, large volume, and high density. Bone can also have disease/disorder-specific challenges to delivery, including sub-micrometer canaliculi, which can serve as a reservoir for bacteria [102,103]. More generally, NP protein adsorption reduces targeting efficacy and shifts biodistribution to the reticuloendothelial system, which contributes to poor bone accumulation [70]. Improvements in surface chemistry to modulate protein adsorption and maintain targeting specificity, which have recently been reviewed elsewhere [104–106], are critical to improve diagnostic/theranostic resolution as well as safety and efficacy.

For local applications, the addition of diagnostic or theranostic capabilities to conventional orthopaedic implants/treatments also has great potential benefit. In particular, augmenting local biomaterials or implants would enable rapid intervention and/or minimize the need for additional invasive procedures. Incorporating sensing functionalities within surgical implants may enable early detection of complications such as poor graft integration, infection, nonunion, or implant loosening. Vascularization and host cell infiltration are important metrics for successful tissue integration/regeneration [107,108] and valuable to inform clinical decision-making regarding need for revision surgery. However, current clinical diagnostics lack resolution to enable imaging of microvessels and cells in vivo [109,110]. Similarly, implant-associated bacterial infections are difficult to detect due to limited specificity of existing diagnostic modalities versus normal post-surgical inflammation [111]. Incorporating bacteria-responsive materials within implants or antibiotic PMMA beads could allow for enhanced infection detection and treatment post-operatively [112]. While these technologies have not yet been developed, locally delivered theranostics represent an important area of exploration in the future.

Diagnostic requirements differ between research and clinical applications. Many of the biomaterials described here have fluorescent detection modalities, which provide unique diagnostic information including bacterial detection [37] and metabolite or protein sensing [16,86]. While efficacious for research purposes, fluorescence is not a standard clinical detection modality and is limited by tissue penetration. Rather, x-rays/CT, magnetic imaging, and ultrasound are clinical mainstays that can be augmented to improve contrast and specificity for orthopaedic applications through development of diagnostic and theranostic biomaterials.

Clinical translation of the orthopaedic diagnostic and theranostic biomaterials discussed here will be challenging. Nanoparticle diagnostic agents have been used clinically for other applications and provide a road map for orthopaedic applications [113]. However, the newness (>95% of publications to date with “theranostics” as a keyword in Web of Science are from 2012 or later) and complexity of theranostics makes their path to regulatory approval unclear. Early theranostics relied on relatively simple radioactive payloads and chelating agents and do not provide good models for the translation of more complex biomaterials approaches necessitated by orthopaedic applications. Nevertheless, for successful translation of orthopaedic diagnostics or theranostics, demonstration of safety and efficacy in large animal models is critical due to the difficulties associated with imaging in bone due to depth, complexity, and size. As the biomaterials field continues to grow, the goal is to integrate the advantages offered through the systems described herein to advance orthopaedic diagnostics and theranostics and ultimately improve patient quality of life.

Conclusions

Early diagnosis and treatment of orthopaedic pathologies is important to treat diseases and injuries effectively and limit undue morbidity. However, traditional diagnostic modalities are subject to limitations, prompting the need for advances in orthopaedic diagnostic and theranostic biomaterials. Bone presents unique challenges for diagnostics and theranostics compared to other tissues due to its unique composition and limited accessibility. Several promising biomaterials strategies have emerged for bone diagnostic and theranostic applications, though work in this area is still relatively new. While current clinically approved applications are limited, the rapid development and expansion of diagnostic and therapeutic modalities within biomaterials offers tremendous promise for clinical use.