Injectable Functional Biomaterials For Minimally Invasive Surgery

REVIEW
www.advhealthmat.de
Injectable Functional Biomaterials for Minimally Invasive

Surgery
Maria Grazia Raucci, Ugo D’Amora, Alfredo Ronca, and Luigi Ambrosio*
minimally invasive surgery, making it a

Injectable materials represent very attractive ready-to-use biomaterials for routine procedure.[3] The growing need for
application in minimally invasive surgical procedures. It is shown that this minimally invasive approaches has led to
approach to treat, for example, vertebral fracture, craniofacial defects, or increased focus on the synthesis and de-
tumor resection has significant clinical potential in the biomedical field. In the velopment of smart injectable biomateri-
als. Injectable biomaterials are materials
last four decades, calcium phosphate cements have been widely used as
that can be injected at room temperature
injectable materials for orthopedic surgery due to their excellent properties in into the site of interest where they solid-
terms of biocompatibility and osteoconductivity. However, few clinical studies ify in response to certain biological condi-
have demonstrated certain weaknesses of these cements, which include high tions. One of the most important require-
viscosity, long degradation time, and difficulties being manipulated. To ments of an injectable biomaterial is the
ability to gel when applied to the tissue,
overcome these limitations, the use of sol-gel technology has been
via chemical reactions due to the mixing
investigated, which has shown good results for synthesis of injectable of two components, changes in pH or tem-
calcium phosphate-based materials. In the last few decades, injectable perature, or even application of light.[4] Sec-
hydrogels have gained increasing attention owing to their structural ond, to be called an injectable, the max-
similarities with the extracellular matrix, easy process conditions, and imum force that must be applied to in-
potential applications in minimally invasive surgery. However, the need to ject the biomaterial should not exceed 100
N.[5] Recently, injectable biomaterials have
protect cells during injection leads to the development of double network
been applied in the field of tissue engi-
injectable hydrogels that are capable of being cross-linked in situ. This review neering as an alternative to implantation
will provide the current state of the art and recent advances in the field of surgery because of their minimally inva-
injectable biomaterials for minimally invasive surgery. sive nature. Injectable materials can fill ir-
regular shape defects and can be easily in-
tegrated with the patient tissue, thereby
accelerating the repair process.[6] These
specific properties of injectable biomaterials can overcome the
1. Introduction
difficulties of cell seeding, cell adhesion, and delivery of therapeu-
Over the past few years, researchers have explored regenera- tic agents, as these compounds can be mixed with the material
tive medicine using a combination of biomaterials and bioac- solution before being injected in situ.[7] Therefore, adequate me-
tive molecules to solve problems associated with age-related chanical and rheological properties, biocompatibility, degradabil-
disease and shortage of tissue and organ donors.[1] Traditional ity, tissue-specific interactions, and nontoxicity of the degradation
therapeutic approaches have a number of disadvantages includ- products are some of the basic requirements that an injectable
ing long hospitalization, highly invasive procedures, anesthesia, material must fulfill to be considered a suitable tissue engineer-
blood transfusions, and prolonged postoperative rehabilitation.[2] ing material. Powdered poly(methyl methacrylate) (PMMA), in
Minimally invasive orthopedic surgery, also known as “keyhole” combination with liquid methyl methacrylate monomer (MMA),
surgery, in combination with smart biomaterials, can be adopted is the most used injectable material in orthopedics.[2a] Although
in both orthopedics and dentistry to reduce the risk of com- various compositions have been tried by mixing PMMA pow-
plications. Progress in tissue engineering, smart biomaterials der with liquid MMA and other ingredients (such as calcium
and new endoscopic techniques has led to great advances in phosphate),[8] these materials suffer from many drawbacks. Non-
degradability, absence of bioactivity, presence of unreacted toxic
monomer, and high curing temperatures are some of the prob-
Dr. M. G. Raucci, Dr. U. D’Amora, Dr. A. Ronca, Dr. L. Ambrosio lems related to the use of PMMA cement in orthopedics.[2a,9] This
Institute of Polymers, Composites and Biomaterials
National Research Council (IPCB-CNR) problem could be partially alleviated by using injectable calcium
Viale J.F. Kennedy 54, Mostra d’Oltremare Pad.20, Naples 80125, Italy phosphate cements (CPCs), which have been extensively used
E-mail: luigi.ambrosio@cnr.it in orthopedic surgery due to their chemical similarity to bone
and their capacity to harden in situ.[10] CPCs have been consid-
The ORCID identification number(s) for the author(s) of this article ered a promising injectable material for bone-defect repair owing
can be found under https://doi.org/10.1002/adhm.202000349
to their peculiar characteristics such as nontoxicity, promotion
DOI: 10.1002/adhm.202000349
Adv. Healthcare Mater. 2020, 9, 2000349 2000349 (1 of 20) © 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com www.advhealthmat.de
of bone ingrowth, bioactive behavior, and capacity to harden in

Maria Grazia Raucci is a re-
situ.[11] However, CPCs also suffer from certain drawbacks re-
search scientist at IPCB-CNR
lated to their brittle nature, slow resorption, fast setting time,
where she leads the Tissue
and noncontrolled porosity that do not allow bone ingrowth.[12]
Engineering & Cell Culture
Though significant research has been carried out to reinforce
Laboratory and associate pro-
CPCs using both synthetic and natural fibers or polymeric addi-
fessor in material science and
tives, very limited reinforcement effect has been achieved.[13] The
technology. She has developed
use of biodegradable polymers and/or composites in the field of
her skills, in addition to those
orthopedics could overcome the main drawbacks of CPCs, ow-
acquired through her Ph.D.
ing to their peculiar properties. First, biodegradable polymeric
program and some years of
bone cements do not have to be removed after use because they
collaboration with the CNR,
can be modified to degrade in situ, and the degradation prod-
through different international
ucts can be naturally metabolized in the body. Second, progres-
experiences. Her expertise in-
sive material degradation at the implantation site promotes bone
cludes in vitro analysis, cell–material interactions, design
regeneration.[2a]
and development of injectable and scaffold ceramics and hy-
Among polymeric materials, synthetic and natural injectable
brid materials by sol-gel. She is national coordinator of the
hydrogels have attracted increasing attention of scientists for ap-
PRIN2017 program and principal investigator of scientific
plication in minimally invasive surgery.[14] Owing to the abil-
collaborations with different biomedical companies and in-
ity of these hydrogels to retain a significant amount of water,
ternational cooperation.
they have found several applications for the controlled deliv-
ery of substances such as nutrients and by-products of cellu- Ugo D’Amora is a researcher
lar metabolism.[15] In fact, hydrogels can entrap water-soluble scientist at IPCB-CNR. In 2009,
drugs and therapeutic cells including stem cells, and due to he graduated cum laude in ma-
their polymeric nature, can be easily biofunctionalized in order terials engineering. In 2013, he
to support more sophisticated functions. In this scenario, the achieved a Ph.D. in materials
progress and advancements in the synthesis and development and structures engineering.
of hydrogels has led to the growth of an innovative class of ma- He was visiting scientist at
terials called “smart injectable hydrogels” with tunable proper- AO Research Institute Davos,
ties triggered by specific stimuli. Natural hydrogels are advan- Switzerland, in 2015/2016 and
tageous owing to the intrinsic presence of bioactive groups that at NERCB – Sichuan University,
can improve cell recognition, adhesion, and proliferation, result- Chengdu, China, in 2017/2018.
ing in a biological repair response.[16] Natural hydrogels are usu- His main research interests
ally based on natural polysaccharides such as alginate, agarose, include material functional-
chitosan, and hyaluronic acid (HA), or derived from proteins ization approaches, design and preparation of polymer and
such as collagen, gelatin, and fibrin. They represent a suitable nanocomposite scaffolds for tissue engineering applications
environment for cells because they closely simulate the biolog- using additive manufacturing techniques, and their charac-
ical properties of the extracellular matrix (ECM).[17] However, terization.
the main drawbacks of natural hydrogels include poor mechan-
ical properties and fast and unpredictable degradation rates that Luigi Ambrosio is director of
strongly depend on the individual, enzyme levels, and injection the IPCB-CNR. He received his
site.[16a,18] Chemical modification of natural hydrogels, such as doctoral degree in chemical
addition of specific functional groups and macromolecules, can engineering from the University
overcome their intrinsic limitations by improving their mechan- of Naples “Federico II”. He is a
ical properties and tuning their biodegradability.[19] Synthetic hy- qualified full professor in bio-
drogels have great benefits in regenerative medicine owing to engineering and in materials
their programmable and reproducible nature.[20] Among syn- science and technology. He is
thetic hydrophilic hydrogels, poly(ethylene glycol) (PEG), poly Fellow of American Institute for
(2-hydroxyethyl methacrylate) (PHEMA), and poly(vinyl alcohol) Medical and Biological Engi-
(PVA) are the most used hydrogels in the biomedical field be- neering, Fellow of Biomaterials
cause of their high hydrophilicity, nontoxicity, and potential to Science and Engineering. Fel-
be easily functionalized by chemical reactions.[21] However, the low of the European Alliance for
lack of biodegradability of these hydrogels has led to the devel- Medical and Biomedical Engineering & Science and Member
opment of several synthetic biodegradable copolymers of PEG of the European Academy of Science (2019).
with poly(lactic-co-glycolic) acid (PLGA) and polycaprolactone
(PCL).[22] Therefore, injectable hydrogels have found applica-
tions not only as fracture fixations in orthopedics but also as
drugs and cell carriers for tissue regeneration because the hy-
drogel matrix can provide mechanical protection to cells during
injection (Figure 1).[1,19a,23] However, most natural hydrogels are
Figure 1. Schematic representation of injectable biomaterials for minimally invasive orthopedic surgery.
usually weak and degrade rapidly under biological conditions.[24] into four different classes based on their chemical nature: acry-
To overcome this drawback, a particular class of interpenetrating late bone cements, calcium phosphate cements, calcium sulfate
network (IPN) polymers, called double network (DN) hydrogels, cements, and composite materials (Table 1).
have been synthesized recently.[25] Gong et al. developed DN hy-
drogels for the first time by using a brittle polyelectrolyte as the
first network and a neutral ductile polymer as the second net- 2.1. Acrylate Bone Cements
work to enhance fracture energy against mechanical stress.[19c,26]
Recently, many authors have attempted to synthesize novel DN For several years, acrylate bone cements played an important
injectable hydrogels by addressing issues such as rapid cross- role in orthopedic surgery.[46] Particularly, one of the most im-
linking, injectability, and cytocompatibility.[27] portant acrylate bone cements is based on PMMA, with differ-
Progress in minimally invasive surgery and related decrease in ent applications from ophthalmology[47] (i.e., intraocular lenses)
patient recovery time represent two key aspects strictly linked to and dentistry[48] (dental fillers) to orthopedics, where it is used
the development of innovative injectable functional biomaterials. in stabilization of prosthesis and repair of vertebral fractures.
Though there are already many reviews on injectable materials The PMMA formula available in the market is based on a liq-
for biomedical applications,[9d,16a,17a,28] this work will summarize uid to solid phase ratio of 2:1. The liquid phase consists of
the state of the art and the most significant advances in functional methyl methacrylate monomers, accelerators, and stabilizers,
biomaterials for application in minimally invasive procedures in- while the powder phase consists of a polymer, catalyst, and radio-
cluding bone, osteochondral, and spine surgery. opacifier (i.e., BaSO4 or ZrO2 ). The polymerization reaction be-
gins with an exothermic reaction of ≈20–30 min after mixing of
the two phases to form nonbiodegradable PMMA with compres-
2. Injectable Bone Cements sive strength ≥ 70 MPa and elastic modulus ≥1800 MPa.[49]
Owing to these peculiar properties, PMMA-based cement is
In recent years, injectable bone cements with self-setting prop- the first Food and Drug Administration (FDA)-approved in-
erties and good handling characteristics have been largely used jectable material used for minimally invasive surgical treatments
for different applications, particularly, bone augmentation proce- such as percutaneous vertebroplasty and kyphoplasty for os-
dures (i.e., orthopedic and maxillofacial surgeries) and bone re- teoporotic vertebral fractures. However, the main drawback of
construction (as fillers of bone cysts). Injectable bone cements PMMA cements is the exothermic polymerization reaction.[50]
possess many important properties such as biocompatibility, In vitro investigations have established that the temperature
bioactivity, osteoconductivity, high radiopacity, low curing tem- changes from 70 °C to 120 °C depending on the volume of ce-
perature, appropriate cohesion, microporosity to allow the flow ments, room temperature, and monomer/polymer ratio. How-
of body fluid, macroporosity for cell infiltration, and appropriate ever, an in vivo animal study demonstrated that the temperature
resorption rate depending on the application site. Independent of reached a value in the range of 40–60 °C, which is the limit for
these properties, injectable bone cements may be differentiated protein denaturation and tissue necrosis.
Table 1. List of some commercially available injectable biomaterials and their composition.
Categories Product Supplier Composition Refs.
Acrylic bone cement CMW1 DePuy Synthes Inc PMMA, MMA, BaSO4 [ 29 ]
Smartset HV PMMA-co-PMA, MMA, ZiO2 [ 30 ]
Endurance PMMA, PMMA-co-PS, MMA, BaSO4 [ 31 ]
Simplex P Stryker Inc PMMA-co-PS, PMMA, BaSO4 [ 32 ]
Spineplex PMMA, PMMA-co-PS, BaSO4 [32a ]
Palacos R Heraeus Inc PMA, MMA, ZiO2 [ 29b,33]
Osteopal V PMMA-co-PMA, MMA, ZiO2 [32b]
KyphX HV-R Kyphon Inc PMMA-co-PS, MMA, BaSo4 [ 34 ]
CPC Norian Srs Synthes Inc MCP, 𝛼-TCP, CaCO3 [ 10,35]
ChronOs inject 𝛽-TCP, MCP, MHPT [ 10,35]
Bone Source Stryker Inc DCP, TTCP [ 36 ]
HydroSet TCP, DCPD, TSC [ 37 ]
Calcibon Biomet Inc 𝛼-TCP, DCP, CaCO3 [ 38 ]
Mimix TTCP, 𝛼-TCP [ 39 ]
𝛼-BSM ETEX Corp ltd ACP, DCPD [ 10 ]
Callos Skeletal Kinetics LLC 𝛼-TCP, MCPM, CaCO3 [ 40 ]
CSC BonePlast Biomet Inc CaSO4 [ 35 ]
MIIG X3 Wright Medical Inc 𝛼-CaSO4 [ 41 ]
CPC-CSC Pro-Dense Wright medical Inc CaSO4 , DCPD, 𝛽-TCP [ 42 ]
Cerament Bone Support AB Inc CaSO4 , HAp [ 43 ]
Composite material Cortoss Orthovita Inc Bis-GMA, Bis-EMA, TEGMA, glass and ceramic fillers [ 32a,44]
CopiOs ZimmerBiomet Inc DCPD, Type I Bovine collagen [ 45 ]
Bisphenol A-glycidyl methacrylate (Bis-GMA), Dicalcium phosphate (DCP), Dicalcium phosphate dehydrate (DCPD), Ethyl methacrylate (EMA), Monocalcium phosphate
(MCP), Monocalcium phosphate monohydrate (MCPM), Magnesium hydrogen phosphate trihydrate (MHPT), Triethylene glycol methyl ether methacrylate (TEGMA),
Trisodium citrate (TSC), Tetracalcium phosphate (TTCP).
Indeed, long-term clinical reviews have demonstrated certain and fast setting time (<10 min).[54] Owing to these properties,
complications associated with the use of PMMA cement, repre- CPCs were approved by the FDA and introduced in clinical prac-
sented by tissue necrosis, bone resorption, and low osseointegra- tice. CPCs may be categorized in different ways, one of which
tion due to nonbioactive and nonbiodegradable nature of the ce- is based on the resorption rate. On the basis of resorption rate,
ment. To overcome these complications, it is necessary to develop CPCs may be classified as apatite and brushite; brushite ce-
new formulations of PMMA cement. ments are biocompatible and bioresorbable, with a resorption
Currently, the only possible modification is the development rate higher than apatite.[55] Apatite cements may form different
of PMMA composites by mixing PMMA with bioactive and apatite products such as calcium-deficient HAp and carbonate-
biodegradable biological compounds. Some examples include apatite. Generally, CPCs are well defined as a blend of one or
the modification of PMMA with bioactive solid signals such as more calcium phosphate powders with a liquid component to
hydroxyapatite micro/nanoparticles (HAp)[51] or chitosan, which form a paste capable of self-hardening in situ and forming a
improved bioactivity, porosity, and osteoconductivity, and re- three-dimensional (3D) structure at the bone defect site.[56] The
duced the setting temperature and elastic modulus.[52] One more hardening is due to a slow exothermic process without a shrink
reformulation was based on the combination of PMMA and reaction. However, there are three main limitations related to
mineralized collagen[53] that improved osteoconductivity, and re- the use of CPCs as injectable materials in minimally invasive
duced the setting temperature and elastic modulus. From the surgery: 1) uncontrolled porosity, 2) separation of solid and liq-
point of view of clinical translation and commercialization of uid phases during injection, and 3) disintegration of paste during
these reformulations, the regulatory risks may be moderated by contact with body fluids.
the medical history of PMMA cements. The phase separation leads to extravasation at the injection
site with a decrease in viscosity and mechanical strength. Fur-
thermore, the disintegration of CPCs in the body leads to an
inflammatory reaction with several effects such as blood clotting
2.2. Calcium Phosphate-Based Cements
and cement embolism. Hence, different strategies have been
explored to improve the injectability of CPCs, which include:
CPCs have potential orthopedic and cranio-maxillofacial appli-
i) development of macroporosity by introducing a foaming
cations because of their unique properties such as bioactivity,
agent, for example, production of injectable macroporous CPC
biocompatibility, biological composition similar to the inorganic
by syringe-foaming method using a silanized hydroxypropyl
component of bone, osteoconductivity, moldability, injectability,
methylcellulose (Si-HPMC);[57] the Si-HPMC improves the Calcium phosphate-based composite materials exhibit enhanced
rheological properties of CPCs, improving its injectability and osteoconductive and mechanical properties as compared to indi-
cohesion; ii) introduction of a polymer (i.e., gelatin, chitosan, vidual materials. However, an important issue to be considered is
cellulose, HA, PVA, etc.) in the liquid phase to increase viscosity; the control of bonding at the ceramic–polymer interface, which
and iii) optimization of solid phase such as particle size and may compromise the mechanical properties. Liu et al. demon-
shape and particle–particle interactions.[58] strated the possibility of producing composite materials based
There are two main mechanisms of CPC degradation: the first on polyethylene glycol/poly(butylene terephthalate) (PEG/PBT)
one is based on passive resorption through chemical dissolution, copolymer and HAp using diisocyanate as the coupling agent. In
while the second one is based on active resorption mediated by this way, it was possible to significantly improve the elastic mod-
cells. Both the processes may be regulated by physical, chem- ulus and strength of the polymer.[64] Moreover, adhesion at the
ical, and biological factors: physical factors are represented by ceramic–polymer interface could also be increased by modulat-
porosity, topography, surface area, and crystalline phase; chem- ing the processing parameters. Similarly, the mechanical perfor-
ical factors are characterized by composition and ionic substitu- mance of composite materials could also be modified by varying
tion (F− , Mg2+ , Na+ , CO3 2− ); biological factors include involve- the organic and inorganic compositions at different temperatures
ment of macrophages. It is possible to improve the degradation and pressures.[64]
process by introducing a porogenic agent such as salt nanopar- Ambrosio et al. have reported the scientific and clinical
ticles or polymeric dense microspheres (such as PLGA), which achievements of a composite cement based on PVA and a spe-
improves degradation after 6 weeks of in vivo implantation as cific formulation of calcium phosphate (H-cem: 𝛼-TCP) (98% wt)
compared to hollow PLGA microspheres.[59] and HAp (2% wt).[58] The presence of an injectable biodegrad-
An important biological property of CPCs is stimulation of able polymer improved the mechanical and biological aspects of
osseointegration. CPCs are particularly reported to be osteo- the cement. In particular, it was demonstrated that the combina-
conductive, but several studies have demonstrated the osteoin- tion of H-cem at a specific L/P ratio produces a ready-to-use prod-
ductive properties of CPCs with the ability to induce new tis- uct with injectability and setting time properties appropriate for
sue formation without osteogenic factors. This property is due clinical application without any adverse reactions to the body. In-
to the combination of different effects such as topography, mi- deed, after 12 weeks of in vivo implantation in the distal femoral
cro/macroporosity, and chemical composition. rabbit condyle, formation of new bone tissue with improved me-
chanical properties was observed. In particular, the PVA/H-cem
composite increased the Vickers hardness degree (HV) of the
2.3. Calcium Sulfate-Based Cements implant from 31.8 ± 5.2 at 4 weeks to 41.6 ± 5.3 at 12 weeks.
Moreover, bone mineralization index (BMI) value of PVA/H-cem
Calcium sulfate (CS)-based cement has a long history of being (74.5 ± 5.7) was higher than that of H-cem (58.6 ± 11.5) at
used for treatment of bone defects due to pain, infection, or 12 weeks.
after cancer removal. In particular, surgical-grade CS-based ce- One more feature to be considered in the design of compos-
ment was developed as 𝛼-CS hemihydrates, which when mixed ite cements is macroporosity for cell infiltration. Some strate-
with water transformed to CS dihydrate, forming a solid or semi- gies allow the creation of macropores in the cement materials.
solid paste.[60] It has been reported that CS-based bone cements For example, Mikos et al. recently developed GMPs/PLGA/CPC
not only have good biocompatibility and osteoconductive prop- composites by incorporating PLGA and glucose microparticles
erties without any inflammatory reaction, but also have rapid (GMPs) in calcium phosphate cements.[65] It has been demon-
self-hardening capacity and biodegradability.[61] Furthermore, 𝛼- strated that initial macroporosity may be obtained by early GMP
CS hemihydrates play an important role in HAp and beta trical- dissolution in a calcium phosphate composite, thus inhibiting
cium phosphate (TCP) to control resorption rate and improve the decrease in pH due to the release of PLGA degradation prod-
osteoconduction. Hsu et al. developed an advanced approach to ucts. Moreover, considering that degradation of PLGA occurs in
produce 𝛼-CS hemihydrate without adding chemical reagents.[62] a few weeks, it was possible to obtain additional macroporosity
In particular, 𝛼-CS hemihydrate with high biocompatibility and in the material at a later time-point of implantation.
angiogenic and osteogenic properties may be obtained by us-
ing a microwave synthesizer with the possibility of controlling
the temperature of the reaction and pressure parameters. Kelly 3. Injectable Materials by Sol-Gel Technology
and Wilkins reported the clinical use of a minimally invasive in-
jectable CS graft in 15 patients with bone defects secondary to The sol–gel approach represents a valid route for synthesis of
traumatic injury.[63] The in vivo results demonstrated that these calcium phosphate (CaP) and composite materials for biomed-
materials induced significant bone healing; and in 14 out of the ical applications.[66] In particular, as this chemical process re-
15 patients, the bone graft was replaced with new bone tissue for- quires room temperature, certain bioactive and/or therapeutic
mation after only 8 weeks. molecules such as growth factors, peptides, dendrimer, antibi-
otics can be introduced during the process. The sol–gel process
includes four steps: i) synthesis of inorganic networks, ii) real-
2.4. Calcium Phosphate-Based Composite Cements ization of colloid solution (sol), iii) gelification of the sol to form
a gel complex, and iv) aging. The chemistry of sol–gel is based
As CPCs exhibit brittle mechanical behavior, introduction of poly- on the hydrolysis and polycondensation reactions of metal alkox-
mer reinforcement has been considered an alternative strategy. ide precursors (Figure 2).[67] This process is influenced by several
Figure 2. Flow chart of the sol-gel method for the synthesis of inorganic and hybrid organo–inorganic CaP.
parameters such as pH, temperature, solvent, and concentration strongly with the GO sheets.[71c] The presence of GO improved
and chemical structure of the precursors. the stability and biological properties in terms of adhesion,
This process is suitable for synthesis of HAp with particle sizes proliferation, and differentiation of hMSCs through osteoblast
ranging from nanometric to micrometric scale, with low crys- phenotype.
tallinity and higher bioactivity. Furthermore, the full injectabil- Sol-gel technology may also be used for development of
ity allows filling of bone and/or dental defects with different organic–inorganic hybrids based on natural and synthetic poly-
sizes and geometries. However, considering their low mechan- mers with calcium phosphate nanoparticles. In this way, the
ical properties (i.e., compressive strength), CaP are limited to functionality of polymers and the bioactivity of the inorganic
few applications such as bone fillers, maxillofacial surgical pro- phase are combined. This co-networking between the phases en-
cedures, and repair of craniofacial defects and dental fillings. hances the performance of hybrids in terms of physicochemical,
To overcome these limitations, the possibility of intro- mechanical, and biological responses.
ducing a reinforcing element such as carbon nanotubes Nowadays, the use of synthetic polymers has become a widely
(CNTs) in strontium-modified calcium phosphate (Sr-CaP) was used alternative strategy in bone tissue engineering. The com-
described;[68] and an injectable composite material with good monly used synthetic polymers are PCL, polyglycolic acid (PGA),
biological properties in terms of adhesion and osteoblast cell PLA, and their corresponding copolymers. However, among the
growth was obtained.[69] It was also demonstrated that modu- tissue-inspired hybrids, PCL and calcium phosphate nanoparti-
lation of CNT topography increased protein adsorption and im- cles have received increasing attention. PCL is a biodegradable
proved proliferation and differentiation of human mesenchymal aliphatic polyester approved by US FDA for craniofacial indica-
stem cells (hMSCs) through osteoblast phenotype[70] by induc- tions, with modulated mechanical properties, slow degradation
ing the expression of specific osteogenic markers (i.e., alkaline rate to allow new tissue formation, and good biocompatibility.[73]
phosphatase, osteopontin, and osteocalcin). Furthermore, car- Dessì et al. demonstrated that PCL/HAp hybrids obtained by
bonaceous materials such as graphene oxide (GO) have also been sol–gel approach showed full injectability and required a lower
used to prepare biomineralized hybrids by using a biomimetic force (7 vs 11 N) for complete extrusion. Indeed, the flow in
approach or sol–gel method.[71] Additionally, it was observed that the syringe completely occurs without phase separation, nee-
addition of multiwalled CNTs to calcium phosphates inhibited in dle disruption, or hardening in the syringe, which is typical
vitro osteoclastogenesis.[72] of CPCs.[74] Another example is represented by a PVA/bioglass
The presence of epoxide, hydroxyl, and carbonyl groups on the 45S5-based composite. This composite exhibited higher tensile
GO plane offers specific reactive sites for the nucleation and crys- strength (26.0 MPa) and higher rate of proliferation of MG63 cells
tallization of calcium phosphate nanoparticles. The enhanced (human osteosarcoma cells; 2.60 fold) as compared to neat PVA
mechanical properties in a reduced state (i.e., chemical interac- (tensile strength, 5.5 MPa; MG63 cell proliferation rate, 1.60) at
tions with calcium or phosphate ions), high hydrophilicity, and day 7, as well as increased ALP enzyme activity (20 U L-1 ) as
good biocompatibility of GO sheets suggest that GO may be a compared to neat PVA (17 U L-1 ). Therefore, this composite pos-
potential nanoscale reinforcement filler for improving bioactivity sessed substantial potential in bone tissue engineering.[75] Owing
and interfacial bonding in the composites. Raucci et al. demon- to their important properties such as biocompatibility, biodegrad-
strated the possibility of obtaining HAp nanocrystals (diameter: ability, and bioactivity, certain natural polymers have been used
5 ± 0.7 nm; length: 70 ± 2.5 nm) intercalated homogeneously and to synthesize hybrid materials via the sol-gel route.[76]
Among natural polymers, great attention has been paid to the ties. For example, Paul et al. synthesized Zn- and Si-modified
polysaccharide chitosan.[77] Chitosan is soluble in acidic solution CPC at different compositions by combining sol-gel process and
(pH < 6) due to protonation of the free NH2 groups. This pH sintering.[84] The in vivo study demonstrated that Zn (6%, w/w)-
dependency offers an appropriate tool for the development of CPC cement was the best candidate to induce new tissue forma-
hybrids using sol-gel method. In addition, the presence of NH2 tion faster (BV/TV: 22.78% at 8 weeks). Meanwhile, implantation
and OH groups provides coupling points for cross-linking silica of Zn (9% w/w)-CPC cement showed significantly delayed bone
(SiO2 ) networks. Thus, hybrids based on chitosan-SiO2 exhibited healing at the same time point.
tailored mechanical properties, degradation rates, and good cell Furthermore, composites containing amorphous calcium
compatibility.[78] Oliveira et al. showed that bioglass composed of phosphate (ACP) or calcium fluoride (CaF2 ) nanoparticles and
60% SiO2 , 36% CaO, and 4% P2 O5 (mol%) induced fast precipi- chlorhexidine showed the highest antimicrobial activity with re-
tation of an hydroxycarbonate apatite layer in vitro, demonstrat- duced metabolic activity of the biofilm as compared to the con-
ing a prospective use in bone tissue engineering.[79] The results trols. Therefore, these materials may have potential applications
highlighted that bioglass nanoparticles obtained using sol–gel in orthopedics and dentistry.[85]
approach exhibit higher bioactivity than microparticles obtained An alternative strategy for the development of antimicrobial
using conventional techniques. Furthermore, the nanoparticles nanoparticles was given by Raucci et al., who developed antimi-
were easily incorporated and dispersed into the chitosan matrix crobial calcium phosphate-based biocomposites by loading im-
using the sol-gel method, obtaining a composite that exhibits idazolium ionic liquids (ILs).[86] By optimizing the IL N-alkyl
bioactive properties of bioglass and elasticity of polymers. chain length, it was possible to concurrently modulate the bi-
Regarding collagen, low immune response, and biocompati- ological response including induction of hMSC differentiation
bility represent the main advantages of type I collagen. However, through the osteoblast phenotype, inhibition of bacterial and fun-
collagen alone does not exhibit osteoinductive properties, but os- gal growth, and reduction of inflammatory response. Particularly,
teoconductivity can be induced by combining collagen with cal- the antimicrobial activity was increased by increasing the length
cium phosphate (CaPO4 ). In particular, type I collagen and HAp of alkyl chain (C4MImCl < C10MImCl < C16MImCl). Using this
were found to enhance osteoblast differentiation and simultane- strategy, it was possible to overcome certain limitations related to
ously induce increased osteogenesis.[80] However, some in vivo the use of antibiotic and antimicrobial nanoparticles.
results demonstrated that the implanted HAp/collagen compos- Another important aspect to be considered during an in vivo
ite had different effects depending on the animal’s age. Indeed, surgical implantation is the activation of the cascade of events
HAp/collagen improved regeneration time of bone defects at 8 involved in inflammatory response. Several studies have estab-
weeks in young animals, but the process was delayed in older lished that a modulated inflammatory reaction is involved in
animals.[81] bone repair.[87] This is the situation regarding the implanta-
tion of bioresorbable biomaterials, where macrophages are in-
volved in material degradation (i.e., bioceramics).[88] It is well
3.1. Injectable Calcium Phosphate-Based Materials with known that macrophages are involved in several biological pro-
Therapeutic Properties cesses, such as recruitment, proliferation, and differentiation of
osteoblasts, through the release of growth factors (i.e., BMP,
The increasing aged population and involvement in competitive FGF, and IGF) and inflammatory interleukins such as inter-
activities have increased the demand for medical implantation. leukin 6 (IL-6) and interleukin 1𝛽 (IL-1𝛽). Yuan et al. have suc-
Medical implantation is generally associated with microbial cessfully prepared injectable Sr-modified calcium phosphate gels
infections; indeed, implant failure is usually related to bac- incorporating branched poly(epsilon-lysine) (PS) dendrons with
terial colonization followed by inflammation, which inhibits third-generation branches exposed to phosphoserine (G3-K PS)
complete osteointegration. Moreover, infections associated with using the sol-gel method.[89] The presence of Sr and G3-K PS pro-
in vivo implants are usually due to biofilm development as moted MC3T3-E1 cell adhesion, proliferation, and differentiation
microorganisms, bacteria, and/or fungi in biofilms are resis- by modulating the release of proinflammatory interleukins, in-
tant to antibiotics and host immune response. In these cases, cluding IL-1𝛽, IL-6, and TNF-𝛼, from macrophages.
prolonged systemic antibiotic therapy may be ineffective.[82] Furthermore, an in vivo study using an ovariectomized rat
Therefore, additional clinical treatments such as debridement, femoral defect model confirmed that Sr and G3-K PS enhance
prosthesis removal, and reimplantation are required, which the osteoinductive properties of calcium phosphate gel materials
increases the cost of healthcare systems. Moreover, several draw- by increasing the BV/TV value from 15% to 30% at 8 weeks post
backs such as low efficacy, systemic toxicity, and high healthcare implantation.
costs are associated with systemic antibiotic administration.[83]
Accordingly, an ideal biomaterial to be implanted in the human
body should possess multifunctional properties including in- 4. Natural and Synthetic Injectable Polymer-Based
duction of new tissue formation and prevention or inhibition Biomaterials
of infection and inflammation. Therefore, the development
of multifunctional materials to be used as injectable fillers in Polymer-based hydrogels are hydrophilic and highly hydrated
minimally invasive surgery with local release of antimicrobial materials, forming a soft swollen 3D network that can be sta-
components represents a great challenge for many scientists. bilized through different types of cross-linking approaches.[15,16]
Many studies have considered the design and production of They are structurally comparable to the ECM. Furthermore,
new biomaterials with osteoinductive and antimicrobial proper- due to relatively mild processing conditions and the possibility
Figure 3. Schematic illustration of natural and synthetic biomaterials employed for the design of semi-IPN, self-healing/SM materials and in situ DN
systems.
to be released following a minimally invasive approach, in- to overcome the limitations of both materials are: i) introduc-
jectable hydrogels have gained great interest in the biomedical tion of bioactive moieties through chemical modification using
field.[17b,90] Natural and synthetic biomaterials have been em- specific functional groups, and ii) development of natural and
ployed for the development of injectable hydrogels with differ- synthetic polymer blends.[94] In this scenario, semi-IPN hydro-
ent properties such as shape memory (SM), self-healing, or im- gels with improved mechanical properties, better biocompati-
proved mechanical properties (IPN and DN) for tissue engi- bility, and unique chemistries have been employed in tissue
neering applications (Figure 3).[28b,91] Natural biomaterials con- engineering.[95] Recently, many studies have been carried out to
tain cell-targeting signals such as RGD (Arg-Gly-Asp), which synthesize biocompatible injectable semi-IPN hydrogels for min-
confer excellent biocompatibility.[92] On the other hand, syn- imally invasive surgery.[19c,26,27b,c]
thetic hydrogels have drawn much attention because their prop- In the following sections, we will summarize the injectable
erties can be certainly tuned, but they may lack biological activ- natural and synthetic hydrogels commonly employed for bone,
ity leading to lower biological response.[93] Common approaches cartilage, and nucleus pulposus (NP) regeneration (Table 2).
Table 2. Overview of the natural and synthetic injectable polymer-based biomaterials for bone, cartilage and NP tissue engineering.
Field of Matrix Classification Composition Main outcomes Refs.

application
Bone HA NATURAL High molecular weight MEHA Increased mechanical stiffness; long-term stability; good [ 96 ]
primary cell survival; excellent osteogenic differentiation

in vitro
MeHA/CaP; Maleated HA/CaP Tunable physicochemical, mechanical and morphological [ 19b,c,97]
properties
Alginate Alginate/Sr-Hap Beneficial effects in bone remodeling and in the treatment [ 98 ]
of osteopenic disorders and osteoporosis

PCLA SYNTHETIC PCLA/bioactive glass Bioactive; Biocompatible, Good osteoconductivity; Good [ 99 ]
osteochondral regeneration; Ductile; Fully injectable;

Easy to handle with short setting time
Cartilage Collagen NATURAL Type II coll./ type I coll. Chondrocyte phenotype maintained; Cartilage-specific [ 100]
ECM secreted promotion

Coll./carbon dot Fully injectability; Increased stiffness; Reduced [ 101]
nanoparticles/genipin degradation rate; Increased chondrogenic

differentiation of bone marrow-derived stem cells
Type II coll./PEG/PEG ether Easy injectability; In situ setting; Good cell transplantation [ 102]
tetrasuccinimidyl glutarate
Type II coll./PEG ether Injectability; Stable structure; Maintenance and promotion [ 103]
tetrasuccinimidyl of viability and chondrocytic properties

glutarate/HA/TGF𝛽1
Gelatin Oxidized dextran/amino Tuned degradation profile; Controlled mechanical [ 104]
gelatin/4-arm PEG-acrylate properties; cytocompatible

HA HA/type I coll. Stimulated chondrocyte proliferation; Enhanced [ 105]
proteoglycan synthesis
High molecular weight HA/fibrin Improved biomechanical and histological properties at 6 [ 106]
months post implantation

Furyl-modified HA/PEG Good mechanical properties; short gelation times; high [ 107]
cell viability/proliferation
HA/chitosan/methacrylated glycol Increased cell proliferation and cartilaginous ECM [ 108]
chitosan production
MEHA Good mechanical properties; Chondrocytic phenotype [ 109]
retention; Promotion of cartilage matrix synthesis in

vitro; Acceleration of healing in vivo
Ethylenediamino or Improved mechanical properties; Controlled hydrolysis [ 110]
amino/octadecyl HA and divinyl

sulfone-functionalized inulin
HA/cellulose nanocrystals Good cell/material interaction [ 111]
Fibrin Fibrin/PEG Induced angiogenesis and in situ neovascularization [ 112]
Fibrin/ECM microparticles/TGF Chondrogenesis of freshly isolated fat pad-derived stromal [ 113]
𝛽3 cells in vivo
Fibrin/alginate particles Improved in vitro biological performance and in vivo [ 114]
vascularization
Alginate Oxidized alginate No inflammatory and oxidative stress reactions [ 115]
CaP with 50% of human umbilical Excellent mechanical properties; Fully injectability [ 116]
cord mesenchymal stem

cells—encapsulating alginate
beads/chitosan/fibers
Oxidized alginate/HA Excellent results after 6 weeks postimplantation [ 108b]
Chitosan Glycol chitosan/benzaldehyde Tunable mechanical properties; Good cell/material [ 117]
capped PEG interaction

Chitosan/gelatin Improved mechanical properties; Controlled [ 14d]
biodegradability; Good biocompatibility

(Continued)
Table 2. Continued.
Field of Matrix Classification Composition Main outcomes Refs.

application
PEG SYNTHETIC PEG/bovine chondrocytes Fully Injectable, no adverse inflammatory response [ 118]
Thiol-ene PEG Promotion of yaline-like engineered cartilage [ 119]
HA/PEG Outstanding load-bearing and shape recovery properties; [ 107b]
Short gelation time; Good cell viability, proliferation and

chondrogenesis
Poly (l-glutamic Adipic dihydrazide-modified Tuned gelation time; Tuned equilibrium swelling; Good [ 120]
acid) poly(l-glutamic acid) mechanical properties

aldehyde-modified
poly(l-glutamic acid)
Hydrazide-modified Good viability of the entrapped cells and cytocompatibility; [ 121]
poly(l-glutamic acid) ectopic cartilage formation; Full injectability; Rapid in

Aldehyde-modified alginate vivo gel formation; Good mechanical stability
Poly(oligoethylene Poly(oligoethylene glycol Good mechanical properties; Good cell vitality [ 122]
glycol methacrylate)/cellulose
methacrylate) nanocrystals
Nucleus Collagen NATURAL Atelocollagen type Ability to resist scaffold volume reduction; Highest [ 123]
polposus II/HA/Aggregan/microbial amount of sGAG; High confined compressive strength

transglutaminase
Coll./LMW HA/PEG ether Fully Injectability; Optimized viscoelastic properties, good [ 123]
tetrasuccinimidyl cell/material interactions

glutarate/chitosan
nanoparticles
Gelatin Photocrosslinked NP like differentiation of adipose stromal cells, activation [ 124]
gelatin/methacrylated HA of the integrin 𝛼v𝛽6-TGF-𝛽1 pathway

HA oxidized-HA/adipic acid NP cell synthesis; mRNA gene expression; good [ 125]
dihydrazide biocompatibility
HYADD3 HYAFF120 Gel-like behavior under dynamic conditions [ 126]
HA-pNIPAM Cell phenotype maintained; ECM generation promotion [ 127]
Fibrin Fibrin/HA/FGF-18 Increased type II coll. and carbonic anhydrase XII gene [ 128]
expression
Alginate methacrylated low viscosity Tunable material properties; Good cells/material [ 129]
alginate interactions
Elastin Elastin/glycosaminoglycan/ Resistant and hydrophilic; Good in vivo results [ 130]
collagen
pHEMA SYNTHETIC pHEMA-co-APMA/graft-PAA Good cell viability; Good cell differentiation; Significant [ 131]
decrease in stiffness and modulus values of cellular

hydrogels in comparison with acellular hydrogels at
both day 7 and 14
PEG PEG/HA/pentosan polysulfate Tuned gelation rate and mechanical strength; Promoted [ 132]
proliferation and chondrogenic differentiation of adult

human bone marrow-derived mesenchymal precursor
cells
4.1. Natural Injectable Polymer-Based Biomaterials these properties can be enhanced by combining synthetic and
natural biomaterials.[134]
Natural polymers (such as collagen/gelatin, HA, fibrin, algi-
nate, chitosan, agarose, and elastin) have been employed for
biomedical applications. Owing to their biomimicry, natural poly- 4.1.1. Collagen and Gelatin-Based Injectable Biomaterials
mers are more cell friendly, allowing improved cell/material
interactions.[133] However, poor structural properties and low res- Collagen is a natural antigenic biomaterial present in skin, lig-
idence time are the two main issues restricting their applicability. ament, bone, and cartilage tissues of the body. Owing to this
To overcome these limitations, polymer chains are often func- reason, collagen has been widely employed to produce scaf-
tionalized with chemically cross-linkable moieties. Furthermore, folds for bone, cartilage, and interface tissue engineering.[100,135]
Type I and type II collagens, embedding chondrocytes, were otal role in different mechanisms concerning chondrogenic
used by Yuan et al. to obtain an injectable hydrogel.[100] The au- differentiation.[138] Therefore, owing to its native properties, HA
thors demonstrated that it was also possible to control the me- has been considered an ideal biomaterial for cartilage tissue
chanical properties of the hydrogel by tailoring the concentra- repair.
tion of collagen (type I). Indeed, the compressive modulus of Indeed, HA mimics the natural tissue to obtain a highly hy-
composite hydrogels increased with an increase in type I colla- drated environment.[133] However, the biomechanical stability of
gen, ranging from ≈2 to 6 kPa at different frequencies, with a HA is low; therefore, HA has been mainly used in combination
very weak dependence on frequency. From a biological point of with stronger polymers to improve its mechanical properties.[109]
view, the cells maintained their morphological features, secret- Liao et al. developed an HA/type I collagen composite hydrogel,
ing cartilage-specific ECM. Lu et al. synthesized an injectable hy- which stimulated chondrocyte proliferation and enhanced pro-
drogel conjugating carbon dot nanoparticles onto the backbone teoglycan synthesis.[105] Similarly, Rampichová et al. highlighted
of collagen using the natural cross-linking agent genipin.[101] that fibrin mixed with high molecular weight HA was found to
Genipin and nanoparticles not only enhanced hydrogel stabil- be a suitable hydrogel for chondrocytes in vitro. Furthermore,
ity (reaching a compressive modulus of 42.8 ± 4.2 kPa; 21 times presence of collagen changed the viscoelasticity and improved
higher than that of the non-cross-linked one, 1.9 ± 0.3 kPa) but the biomechanical properties of the hydrogel in vitro. Collagen-
also induced the differentiation of bone marrow-derived stem based structures were clearly characterized by a 25 % higher
cells into chondrogenic phenotype. Similarly, an injectable scaf- Young’s modulus (0.544 ± 0.008 MPa) than noncollagenous
fold was designed by Funayama et al. by employing collagen type ones (0.445 ± 0.005 MPa). However, the risks related to the use
II.[102] The chondrocyte-laden hydrogel was injected into dam- of collagen are still high. Therefore, the authors proposed the use
aged rabbit cartilage. It was observed that the gel bonded to the of a composite fibrin-based hydrogel in combination with high-
adjacent cartilage and bone within several minutes. The results MW HA, as a suitable alternative, which exhibited appropriate
of in vivo tests highlighted good cartilage regeneration capacity biomechanical properties for implantation, promoting ECM
of the hydrogel. production.[106] Furthermore, the gels were also characterized
Using a combination of type II collagen and HA, Kontturi by good cell delivery capabilities.[105,106,139] Yu et al. synthesized
et al. designed an injectable hydrogel capable of forming a stable an injectable HA/PEG hydrogel with improved biomechanical
structure directly in situ for cartilage repair and regeneration.[103] features for cartilage regeneration,[107a] and the embedded cells
Furthermore, the hydrogel was enriched in chondrocytes and demonstrated high viability and proliferation. Considering
chondrogenic growth factors, and the cells embedded in the the benefits of chitosan, including biocompatibility, structural
hydrogel were viable. Halloran et al. showed that a type II atelo- resemblance to glycosaminoglycan, and the capability to easily
collagen scaffold based on different concentrations of aggrecan form ionic complexes, Park et al. designed a chondrocyte-laden
and HA, cross-linked by microbial transglutaminase (mTGase), injectable hydrogel based on chitosan, HA, and methacry-
was a good cell-laden formulation for NP regeneration.[123] Gloria lated glycol chitosan.[108a] This hydrogel allowed proliferation
et al. demonstrated that a biomaterial synthesized by combining of chondrocytes with increasing deposition of cartilaginous
collagen and molecular weight HA-PEG ether tetrasuccinimidyl ECM.
glutarate, loaded with chitosan nanoparticles, could be used as To overcome its poor mechanical stability and rapid hydrolytic
an injectable hydrogel for NP repair/regeneration.[126] degradation, HA is usually chemically functionalized.
Gelatin is a biodegradable and biocompatible natural pro- Nettles et al. synthesized an in situ photo-cross-linkable HA
tein obtained by the degradation of collagen. Gelatin has a high scaffold for articular cartilage repair. Chondrocytes were encap-
biodegradation rate and poor mechanical stability due to enzy- sulated in cross-linked MEHA and evaluated for their ability to
matic digestion and high solubility under physiological condi- synthesize cartilaginous matrix in vitro. The gels showed com-
tions. However, due to the presence of different chemical groups pressive and dynamic shear moduli of 0.6 and 0.3 kPa, respec-
(NH2 and COOH), many chemical strategies may be employed tively, and a diffusion coefficient of 600–8000 m2 s-1 depending
to increase the residence time of gelatin.[136] Geng et al. pro- on the molecular weight.[109]
duced an injectable hydrogel through a two-step procedure using MEHA gels for hard tissue engineering were developed by
amino gelatin, oxidized dextran, and four-arm PEG-acrylate.[104] Poldervvart et al. The authors used these injectable materials for
The resulting hydrogel exhibited interesting mechanical proper- 3D bioprinting of porous and custom-made scaffolds.[96] In hy-
ties, biodegradability, and biocompatibility. drogels containing a higher concentration of MEHA polymer,
For intervertebral disc repair, a photo-cross-linked MSCs were able to spontaneously differentiate without further
gelatin/methacrylated HA (MEHA) hydrogel capable of in- osteogenic stimuli.
ducing differentiation of adipose stromal cells into NP cells was Similarly, MEHA and maleated HA (MAHA) were investi-
developed by Chen et al.[124] gated for application as injectable photo-cross-linkable systems.
Furthermore, the development of nanocomposite hydrogels by
in situ biomineralization of both MEHA and MAHA polymers
4.1.2. Hyaluronic Acid-Based Injectable Biomaterials was also proposed. In particular, by using a sol-gel approach,
nanocomposite hydrogels with improved physico-chemical
HA, present in the ECM of adult cartilage, is a linear polysac- and mechanical properties for soft/hard tissue regeneration
charide composed of disaccharide units of glucuronic acid were developed.[19b,c,97] Palumbo et al. designed a hydrogel
and N-acetylglucosamine.[137] HA interacts with chondrocytes based on pendant ethylenediamino or amino/octadecyl HA
via the surface receptors CD44 and RHAMM, playing a piv- and divinyl sulfone-functionalized inulin.[110] The resulting
hydrogel exhibited improved mechanical properties (elastic mod- 4.1.4. Alginate-Based Injectable Biomaterials
ulus, 14.8 ± 0.6 kPa; strain, 10%) and decreased susceptibility to
hydrolysis. Such initial mechanical resistance was comparable to Alginate is a polysaccharide derived from brown algae[144] con-
other materials already proposed as scaffolds for chondrocytes, sisting of glucuronic and mannuronic acids. It is one of the
showing an initial elastic modulus between 10 and 50 kPa.[140] most used materials for designing injectable formulations in car-
Furthermore, bovine chondrocytes embedded in this hydrogel tilage tissue engineering owing to its nonimmunogenicity and
were viable and proliferated. Domingues et al. developed a rein- nontoxicity.[144a]
forced HA injectable hydrogel using cellulose nanocrystals.[111] Balakrishnan et al. produced an oxidized alginate-based in-
The biological behavior of the hydrogel was assessed using jectable hydrogel.[115] The hydrogel showed good integration with
human adipose-derived stem cells (hADSCs). The overall re- the cartilage tissue without inducing inflammatory and oxidative
sults showed good cell/material interactions with improved stress reactions. Furthermore, results from biological analyses
proliferation and spreading of cells within the gel. With regard suggested that this hydrogel is a promising injectable formula-
to NP regeneration, oxidized-HA/adipic acid dihydrazide (oxi- tion for cartilage tissue engineering.
HA/ADH) injectable hydrogel was recently developed by Su et al. However, the main limitation of using an injectable alginate
The oxi-HA/ADH hydrogel exhibited a change in its behavior hydrogel is its poor mechanical properties that do not ensure
from a liquid to a gel-like state in situ within a few minutes. Im- structural shape and maintenance of the regenerated tissue. For
portantly, oxi-HA/ADH8 hydrogel allowed NP cell synthesis and this reason, alginate is usually chemically modified or blended
mRNA gene expression and exhibited good biocompatibility.[125] with other biomaterials to improve its mechanical and biological
Similarly, HYADD3, a dodecylamide, and HYAFF120, a cross- behavior.[145]
linked ester of HA, have shown interesting results in terms of For example, Zhao et al. designed an injectable calcium phos-
appropriate NP biological behavior.[141] phate containing 50% of human umbilical cord mesenchymal
In spine research, thermoreversible hydrogels showed great stem cell-encapsulating alginate beads.[116] The incorporation
potential as they provide good injectability and exhibit a of chitosan and fibers increased the strength and toughness
slight gelling mechanism. Peroglio et al. synthesized a cy- of the stem cell construct, reaching an elastic modulus of
tocompatible thermoreversible HA-pNIPAM as an NP cell 0.7 GPa, much higher than that of cancellous bone (0.3 GPa),
carrier.[127] without compromising the injectability, as compared to cal-
cium phosphate-microbeads without fibers.[146] The injectable,
biodegradable, oxidized alginate/HA hydrogel prepared by
Park and Lee showed significant cartilage regeneration at 6
4.1.3. Fibrin-Based Injectable Biomaterials weeks postimplantation.[108b] In another study, a biocompatible
and biodegradable alginate-based hydrogel blend was synthe-
Fibrin is a natural fibrous protein involved in coagulation mech- sized using alginate and O-carboxymethyl chitosan and fibrin
anism and is commonly employed to improve cell attachment, nanoparticles.[147] Jeon et al. produced a possible solution for
proliferation, and differentiation.[142] Fibrin has been used either disc regeneration; they methacrylated low-viscosity alginate,
alone or in combination with other biomaterials to obtain scaf- allowing the production of a photo-crosslinkable hydrogel
folds for cartilage repair/regeneration.[143] Benavides et al. syn- with tunable material properties and good NP cell/material
thesized an injectable hydrogel capable of inducing angiogenesis interactions.[129] Barbosa et al. developed an injectable system
and in situ neovascularization using fibrin hydrogels, PEG, and based on an alginate matrix cross-linked in situ with Sr and re-
human amniotic stem cells.[112] inforced with ceramic Sr-rich microspheres. They demonstrated
Similarly, fibrin hydrogel coupled with cartilage ECM mi- the beneficial role of Sr in bone remodeling and treatment of
croparticles and transforming growth factor-𝛽3 (TGF-𝛽3) has osteoporosis.[98]
been reported to be an alternative beneficial approach for regen-
erating articular cartilage.[113] This hydrogel could induce carti-
lage repair and regeneration. Hwang et al. developed a hybrid 4.1.5. Chitosan-Based Injectable Biomaterials
hydrogel using alginate particles and a fibrin matrix. The inclu-
sion of these particles improved biological performance in vitro As reported previously, chitosan is one of the most abundant
and vascularization in vivo.[114] Indeed, it was observed that the polymers. It is a linear amino polysaccharide composed of re-
dense structure of fibrin hydrogels limited cell spreading, pro- peating units of glucosamine and N-acetyl glucosamine units
liferation, and ECM production, while the porous and fibrillar linked by 𝛽-(1-4) glycosidic bonds; chitosan is obtained by alka-
structure of alginate/fibrin hydrogel better supported cellular in- line deacetylation of chitin, which is structurally present in the ex-
teractions with hydrogel materials. Moreover, mechanical charac- oskeletons of crustaceans.[148] Over the past few years, chitosan
terization of these materials showed a compression modulus of has emerged as a good candidate to restore the properties and
1.30 ± 0.51 kPa, which was significantly lower than that of neat functionalities of cartilage and intervertebral discs.
fibrin and alginate hydrogels (9.14 ± 3.47 and 4.02 ± 2.58 kPa, Recently, Jia et al. investigated the use of rabbit synovial fluid
respectively). Hackel et al. investigated the effects of fibrin–HA mesenchymal stem cells (rbSF-MSCs) embedded in a chitosan
hydrogel and fibroblast growth factor 18 (FGF-18) on NP regen- hydrogel to repair a cartilage defect in rabbits. The results in-
eration. Embedded human NP cells increased collagen type II dicated that the mechanical properties of the hydrogel could
and carbonic anhydrase XII gene expression after 14 and 7 d of be fine-tuned by suitably changing the concentrations of gly-
culture, respectively.[128] col chitosan and benzaldehyde-capped PEG. Further biological
investigations highlighted the good biocompatibility of the hy- tested it in vivo using a nude mouse model. Histological studies
drogel to rbSF-MSCs.[117] highlighted the formation of neocartilage without any adverse
A strong chitosan-gelatin hydrogel was produced by Shen inflammatory response.[118] In a study by Aho et al., an injectable
et al. through an in situ precipitation approach.[14d] This hydro- composite of poly(𝜖-caprolactone-co-d,l-lactic acid) (PCLA) and
gel showed controlled biodegradability and good biocompatibil- bioactive glass S53P4 was used as synthetic bone filler in a rabbit
ity. Furthermore, owing to the electrostatic interactions between cancellous and cartilaginous subchondral defect model.[99] The
chitosan and gelatin, the hydrogels showed improved mechanical composite structure provided an interface with the host tissue
properties with a Young’s modulus of 10.24 ± 1.05 MPa, higher with good osteoconductivity. PLA is an aliphatic biocompatible
than that of human nasoseptal cartilage, articular cartilage, and and biodegradable polyester that has been widely employed
knee meniscus (0.8 MPa, 0.31–0.85 MPa, and 0.17–0.35 MPa, because its degradation products can be naturally metabolized
respectively).[149] in vivo.[154] Yan et al. proposed a poly(l-glutamic acid)-based
injectable hydrogel that showed in vivo gelation and optimized
mechanical and biological properties.[120] Skaalure et al. devel-
oped a chondrocyte-laden degradable hydrogel based on PEG,
4.1.6. Elastin-Based Injectable Biomaterials
which showed promising results for cartilage regeneration.[119]
De France et al. designed a nanocomposite hydrogel based on
Soft tissues including skin, blood vessels, and lungs are mainly
poly(oligoethylene glycol methacrylate) and cellulose nanocrys-
characterized by the presence of elastin. Elastin is an insolu-
tals, which exhibited enhanced mechanical properties. In
ble polymeric protein that confers elastic properties to natural
particular, the inclusion of only 5 wt% cellulose nanocrystals re-
tissues.[150] For this reason, it has been widely employed in car-
sulted in a dramatic enhancement in the mechanical properties
tilage tissue engineering.[151] Fathi et al. produced an injectable
(up to 35-fold increase in storage modulus).[122]
elastin-based hydrogel with tunable gelation kinetics, improved
However, compared to natural biomaterials, synthetic bioma-
structural stability, and high biocompatibility.[23a] A chemically
terials lack biological activity. In order to overcome this problem,
stabilized elastin–glycosaminoglycan–collagen composite hydro-
synthetic biomaterials are modified or combined with bioactive
gel was synthesized by Mercuri et al. to reproduce the resilient
polymers. For example, Yan et al. fabricated various injectable
and hydrophilic nature of NP. Indeed, by blending soluble elastin
poly(l-glutamic acid)/alginate hydrogels by self-cross-linking
aggregates, chondroitin-6-sulfate, HA, and collagen, it was pos-
hydrazide-modified poly(l-glutamic acid) and aldehyde-modified
sible to obtain a resistant and hydrophilic hydrogel capable of
alginate.[121] These injectable hydrogels showed fascinating
restoring its original dimensions and water content following
properties. In addition, Yu et al. produced two HA/PEG-based
multicycle mechanical compression. Preliminary in vitro stud-
injectable hydrogels with good biomechanical stability using
ies involving the use of human hADSCs demonstrated that the
the Diels–Alder click reaction.[107b] By simply tuning the furyl-
hydrogel was cytocompatible and was able to stimulate the differ-
to-maleimide molar ratio and the substitution degree of the
entiation of hADSCs into an NP cell-like phenotype. In vivo bio-
furyl group, the compressive modulus ranged from 4.86 ± 0.42
compatibility studies highlighted interesting results at 4 weeks
to 75.90 ± 5.43 kPa. Moreover, the HA/PEG hydrogel showed
postimplantation.[130]
outstanding load-bearing and shape recovery properties even
after 2000 loading cycles, mimicking the mechanical properties
and behavior of articular cartilage. Furthermore, encapsulated
4.2. Synthetic Injectable Polymer-Based Biomaterials cells exhibited high proliferation, indicating great potential in
cartilage tissue engineering.
Synthetic polymers have been used as injectable biomaterials In spine research, certain synthetic gels showed great capa-
owing to their tunable physicochemical properties. They have bility to attract and differentiate hMSCs into NP phenotype,
a tailored matrix structure and chemical composition and can even without the use of growth factors. An injectable hydrogel
be easily conjugated with growth factors. Moreover, the cross- based on human MSC-laden poly(hydroxyethylmethacrylate)
linking density, mechanical properties, and degradation rate of (pHEMA)-co-aminopropyl methacrylamide (APMA)-graft-
synthetic polymers can be suitably adjusted considering the spe- poly(amidoamine) (PAA) showed a similar property under
cific needs.[152] hypoxic conditions.[131] Frith et al. developed an enzymatically
To date, various synthetic polymers have been studied for cross-linked HA-PEG hydrogel.[132a] The gel was able to stimu-
the development of injectable hydrogels for tissue engineer- late differentiation of undifferentiated mesenchymal precursor
ing. These polymers include PEG, PVA, PLA, poly(propylene cells into chondrocyte-like cells, capable of producing ECM
fumarate) (PPF), poly(propylene fumarate-co-ethylene glycol) components found in NP.[132b]
(PPF-co-EG), poly(l-glutamic acid), 𝛼,𝛽-poly-(N-hydroxyethyl)-dl-
aspartamide, PEG-pNIPAM, methoxy polyethylene glycol, and
methoxy polyethylene glycol–PCL.[7,90b,153] 5. Recent Advances
Among these polymers, PEG has obtained FDA approval
for various biomedical applications. It is a photo-crosslinked 5.1. Injectable Shape Memory and Self-Healing Hydrogels
polymer characterized by hydrophilic properties. PEG can be
chemically functionalized and cross-linked by UV exposure. In the previous sections, we discussed the important classes of
Sims et al. developed an injectable biomaterial based on bovine injectable hydrogels from commercial inorganic CPCs to the
chondrocytes and PEG for cartilage tissue engineering and most innovative injectable functionalized hydrogels. We high-
lighted the most relevant applications focusing our attention on surfactant in the hydrogels.[165] The hydrogel exhibited complete
minimally invasive surgery for bone, cartilage, and NP. However, shape recovery owing to the hydrophobic blocks of the polymer
the application of hydrogels in minimally invasive surgery has acting as physical entanglement and switching segments, above
certain drawbacks related to gelling time, mechanical compatibil- and below the melting temperature (Tm ), respectively.[165]
ity with the surrounding tissue, and the tissue-level organization Kurt et al. reported an innovative method to produce high-
required to obtain immediate functionality.[155] Furthermore, the strength hydrogel with self-healing and SM properties using a
development of mechanical damage and cracks within the phys- noncovalent approach and bulk photopolymerization.[166] They
iological environment does not allow these hydrogels to perform used hydrophobic interactions to generate 3D hydrogels based
their function over a long period of time.[156] To overcome this on linear poly(N,N-dimethylacrylamide) (PDMA) or PAAc chains
drawback, an alternative approach is required to reproduce the containing methacrylate units with long alkyl side chains.[166] The
functional and structural properties of the surrounding tissue hydrogels exhibited high fracture energy of 20 ± 1 kJ m-2 and
and enable the hydrogels to restore their function after me- Young’s modulus up to 308 ± 16 MPa combined with thermally
chanical damage.[157] The development of SM and self-healing induced SM and self-healing properties.
hydrogels represents an innovative approach to prevent the col- Chitosan is usually combined with various chemical compo-
lapse of surrounding tissue and subsequently restore the original nents, such as glycerol phosphate and hydroxyethyl cellulose as
morphology at the implanted site by maintaining cell viability cross-linking agents, to produce stimuli-responsive systems.[167]
and tissue function (Figure 3).[4a,158] Generally, the mechanism Alinejad et al. investigated the potential of different thermosen-
behind the self-healing capability of polymeric hydrogels con- sitive chitosan hydrogels to mimic the mechanical properties of
sists of the following two steps: i) interdiffusion of long polymer NP tissue. These hydrogels were prepared by mixing chitosan
chains through the damaged surfaces and ii) formation of and varying concentrations of three gelling agents: sodium hy-
reversible bonds between the polymer chains.[159] Zhang et al. drogen carbonate, beta-glycerophosphate, and phosphate buffer.
for the first time, prepared SM hydrogels with improved tensile The results showed that the formulation containing 2% chi-
strength (2.3 MPa) using poly(N,N-dimethylacrylamideco-stearyl tosan, 7.5 × 10-3 m sodium hydrogen carbonate, and 0.1 m beta-
methacrylate).[160] Similarly, Gulyuz and Okay developed self- glycerophosphate was the most promising hydrogel for inter-
healing hydrogels with high tensile strength (0.7−1.7 MPa) and vertebral disc regeneration as it exhibited mechanical properties
elongation at break (800−900%) by micellar copolymerization similar to human NP tissue and stimulated synthesis and reten-
of acrylic acid (AAc) with 2 mol% stearyl methacrylate (C18) tion of proteoglycans from NP cells.[168] However, chitosan hy-
as the hydrophobic comonomer in an aqueous NaBr solution drogel could not be prepared under physiological conditions be-
of cetyltrimethylammonium bromide (CTAB).[161] However, cause chitosan could only be dissolved in a solution of acetic acid.
thermally induced SM hydrogels have limited applications in the Liu et al. solved this problem by developing a self-healing and
biomedical field owing to the use of heat to trigger the original injectable polysaccharide hydrogel based on hydroxypropyl chi-
shape.[162] Moreover, most of the SM hydrogels were based on co- tosan (HPCS), cellulose acetoacetate (CAA), and amino-modified
valently cross-linked polymers that provide only SM capabilities cellulose nanocrystals (CNC-NH2 ); HPCS is soluble in water and
without self-healing properties.[163] To address these problems, therefore, this hydrogel could be prepared under physiological
Meng et al. developed a self-healable SM supramolecular hy- conditions. They evaluated the effect of CNC-NH2 on the me-
drogel using phenylboronic acid-grafted alginate (Alg-PBA) and chanical properties and internal morphology of the hydrogel.[169]
PVA on the basis of a reversible PBA–diol ester bond.[162] The Moreover, the materials exhibited pH-responsive properties and
combination of supramolecular interactions and host–guest in- excellent stability under physiological conditions.
teractions provided the hydrogel self-healing and SM properties Oh et al. designed and synthesized a double thermoresponsive
regulated by redox stimuli.[162] Similarly, Miyamae et al. devel- gelatin-based hydrogel stabilized by gelatin-conjugated poly(N-
oped supramolecular hydrogels based on two different kinds isopropyl acrylamide) (pNIPAM).[170] This hydrogel was able to
of guest moieties with different functional properties by using promote fibroblast viability and proliferation.
two kinds of host–guest inclusion complexes to bind polymers A promising platform for tissue engineering applications was
together. The first complex was based on 𝛽-cyclodextrin (𝛽CD) offered by Clarke et al., who synthesized pentapeptide self-
and adamantane (Ad), and the second was based on 𝛽CD and fer- assembled hydrogels characterized by tunable morphology, pH-
rocene (Fc).[164] In this way, the redox-responsiveness of 𝛽CD-Fc responsive gelation mechanism, robust mechanical properties
provides SM properties, while the host–guest complexation en- (with stiffness spanning two orders of magnitude from 2 to
ables self-healing.[164] Despite the development achieved in the 200 kPa by altering the concentration and charge distribution
field of SM/self-healing hydrogels, many applications require of the peptide sequence), as well as shear-thinning and self-
SM materials that can be melt-processed as thermoplastics. healing characteristics. Owing to the presence of non-covalent
However, owing to the covalent cross-linked network, most SM interactions, these hydrogels were able to reform after deforma-
materials cannot be used in typical processing methods such as tion and recover their mechanical properties after application of
injection molding.[161] high strains.[171]
Bilici et al. developed melt-processable SM hydrogels with self- An injectable thermoresponsive hydroxypropyl guar-graft-
healing ability through a supramolecular approach. The hydrogel poly(N-vinylcaprolactam) copolymer was synthesized by
consisted of poly(acrylic acid) (PAAc) network chains containing Parameswaran-Thankam et al.[172] Poly(N-vinylcaprolactam)
20−50 mol% crystallizable n-octadecyl acrylate (C18A) segments is characterized by a phase transition temperature of 33 °C, sim-
together with surfactant micelles. The key idea of this approach ilar to that of pNIPAM; it is biocompatible but lacks bioactivity
was to exclude chemical cross-links and include an extractable and can induce an inflammatory response.[173] For this reason,
a plant-derived polymer (hydroxypropyl guar gum) was also subsequently implanted in mouse models, and improved resis-
employed using methods such as graft polymerization. The graft tance to degradation and optimal tissue response was observed.
copolymer showed excellent thermogelling and injectable prop- Moreover, the DN hydrogel was not only mechanically strong but
erties suitable for in vitro osteoblast differentiation. A chemically also capable of cross-linking within a short reaction time (tgel ).
cross-linked hydrogel containing nano-HAp was developed to This makes it a promising injection-deployable in situ gelable
improve the mechanical strength of the hydrogel.[172] material for application in minimally invasive surgery. Similarly,
Wu et al. designed chitosan/silk fibroin/glycerophosphate Cai et al. designed an injectable hydrogel using two different
composite hydrogels, embedded with copper-containing bioac- physical cross-linking mechanisms.[27c] The first ex vivo cross-
tive glass nanoparticles, to prepare injectable biomaterials for linking step ensured mechanical protection to the cell during in-
cell-free bone repair. They showed full injectability and thermally jection and was based on peptide-based molecular recognition.
triggered in situ gelation at physiological temperature and pH. The second step occurred in situ to reinforce the network and ex-
In vitro studies revealed that these gels supported the growth tend material degradation and cell retention time.[27c] The over-
of seeded MC3T3-E1 and human umbilical vein endothelial all results demonstrated that in situ formation of a DN hydro-
cells, and subsequently induced osteogenesis and angiogenesis, gel could greatly improve the potential of regenerative medicine
respectively.[174] therapies. Yan et al. also developed a DN hydrogel using a com-
bination of glycol chitosan and di-benzaldehyde capped PEG as
the first network and calcium alginate as the weak cross-linked
5.2. Injectable Double-Network Hydrogels second network.
Thus, the authors were able to significantly improve the cyto-
Recently, the possibility of using injectable hydrogels as cell carri- compatibility and mechanical properties of the hydrogel, mak-
ers and carriers for drug delivery has gained increasing attention ing it a possible candidate for the engineering of soft tissues
owing to their biocompatibility and carrier properties.[25b] How- such as articular cartilage.[25b] Liu et al. developed an injectable
ever, the process conditions during sol-gel transition must be thermoresponsive hydrogel composed of xanthan gum (XG) and
mild to ensure the viability of the entrapped cells.[25b,27a] In partic- methylcellulose (MC), and used body temperature for the second
ular, this transition occurs via physical or chemical cross-linking. cross-linking step.[178] The first network was based on the XG
Physical crosslinks include entanglements, hydrogen bonding, double helical strand structure, which makes the XG/MC blend
and hydrophobic interactions. They do not lead to a permanent a highly viscous solution at room temperature. When the tem-
network but are sufficiently strong to prevent solubility of the ma- perature was raised from room temperature to 37 °C, the MC
terials in aqueous media.[175] In contrast, chemical cross-links are cross-links caused gelation of the XG/MC solution. Although re-
permanent and irreversible due to the presence of covalent bonds cent progress has been made in the development of DN in situ
between polymeric chains.[19b] However, these hydrogels are still cross-linked hydrogels, the synthesis of these particular classes
characterized by fast degradability and weak mechanical proper- of materials for application in minimally invasive surgery is still
ties independent of physical or chemical cross-links.[19c,97] challenging.
The development of an in situ DN hydrogel is an elegant and
efficient strategy to improve the mechanical behavior of hydro-
gels. DN hydrogels are defined as semi-IPN polymer networks 6. Conclusions and Future Trends
obtained by the combination of a highly cross-linked rigid first
network and a ductile second network. This particular structure In summary, injectable biomaterials, either natural or synthetic,
leads to an improvement in the fracture energy against mechan- are designed from cross-linked networks in situ for applica-
ical stress.[19c,26,27b,97] tion in minimally invasive surgery. The physicochemical and
Currently, the fabrication of DN hydrogels requires a sequen- mechanical properties of a biomaterial strongly influence its
tial polymerization method where the secondary network usually capability to be used as an injectable system for specific tissue
presents toxic components, such as small cross-linker molecules, repair. Advances in the field, in addition to decreasing patient
that could elicit a significant inflammatory response at the in- discomfort and costs, will also provide new tools for minimally
jection site.[26,176] Orthogonal-click reaction and multistep pho- invasive surgery. However, various alternatives such as the use
topolymerization have been recently used to overcome this lim- of composite materials and natural/synthetic copolymers have
itation, but they strongly compromise the injectability of the proven to be suitable for tissue engineering applications. The
hydrogel.[176,177] Rodell et al. developed, for the first time, a novel development of injectable systems composed of cells, biomate-
self-healing DN system based on chemically modified HA, which rials, and biomolecules that can be injected inside the body to
combines injectability, rapid self-healing, and tunable properties, regenerate different tissues and organs such as bone, cartilage,
using simultaneous interpenetration and cross-linking mecha- and intervertebral disc presents a serious challenge. In this
nisms of the two networks.[27a] scenario, hydrogels can be potential candidates owing to their
In order to eliminate low biocompatible synthetic polymers ability to encapsulate, manipulate, and transfer growth factors
and potentially cytotoxic small molecules such as cross-linkers, and/or bioactive molecules to the surrounding tissue. However,
Zhang et al. developed DN hydrogels based on chemically mod- the low mechanical properties and high degree of degradation
ified chitosan and dextran. An interpenetrating DN structure, of natural hydrogels led to the synthesis of more tough materials
based on thiolated chitosan (Chitosan-NAC) and oxidized dex- such as interpenetrating DN hydrogels. Interpenetrating DN
tran (Odex), was synthesized via Schiff base formation and disul- hydrogels appear to be a great revolution in this field because
fide bond inter-cross-linking.[27b] The synthesized hydrogels were it is possible to tune both the mechanical strength and degra-
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Injectable Functional Biomaterials For Minimally Invasive Surgery

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Injectable Functional Biomaterials For Minimally Invasive Surgery

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REVIEW

Injectable Functional Biomaterials for Minimally Invasive

minimally invasive surgery, making it a

of bone ingrowth, bioactive behavior, and capacity to harden in

Categories Product Supplier Composition Refs.

Smartset HV PMMA-co-PMA, MMA, ZiO2 [ 30 ]

Endurance PMMA, PMMA-co-PS, MMA, BaSO4 [ 31 ]

Simplex P Stryker Inc PMMA-co-PS, PMMA, BaSO4 [ 32 ]

Spineplex PMMA, PMMA-co-PS, BaSO4 [32a ]

Palacos R Heraeus Inc PMA, MMA, ZiO2 [ 29b,33]

Osteopal V PMMA-co-PMA, MMA, ZiO2 [32b]

KyphX HV-R Kyphon Inc PMMA-co-PS, MMA, BaSo4 [ 34 ]

CPC Norian Srs Synthes Inc MCP, 𝛼-TCP, CaCO3 [ 10,35]

ChronOs inject 𝛽-TCP, MCP, MHPT [ 10,35]

Bone Source Stryker Inc DCP, TTCP [ 36 ]

HydroSet TCP, DCPD, TSC [ 37 ]

Calcibon Biomet Inc 𝛼-TCP, DCP, CaCO3 [ 38 ]

Mimix TTCP, 𝛼-TCP [ 39 ]

𝛼-BSM ETEX Corp ltd ACP, DCPD [ 10 ]

Callos Skeletal Kinetics LLC 𝛼-TCP, MCPM, CaCO3 [ 40 ]

CSC BonePlast Biomet Inc CaSO4 [ 35 ]

MIIG X3 Wright Medical Inc 𝛼-CaSO4 [ 41 ]

CPC-CSC Pro-Dense Wright medical Inc CaSO4 , DCPD, 𝛽-TCP [ 42 ]

Cerament Bone Support AB Inc CaSO4 , HAp [ 43 ]

CopiOs ZimmerBiomet Inc DCPD, Type I Bovine collagen [ 45 ]

Field of Matrix Classiﬁcation Composition Main outcomes Refs.

primary cell survival; excellent osteogenic diﬀerentiation

of osteopenic disorders and osteoporosis

osteochondral regeneration; Ductile; Fully injectable;

ECM secreted promotion

nanoparticles/genipin degradation rate; Increased chondrogenic

tetrasuccinimidyl of viability and chondrocytic properties

gelatin/4-arm PEG-acrylate properties; cytocompatible

months post implantation

retention; Promotion of cartilage matrix synthesis in

amino/octadecyl HA and divinyl

Fibrin Fibrin/PEG Induced angiogenesis and in situ neovascularization [ 112]

Fibrin/ECM microparticles/TGF Chondrogenesis of freshly isolated fat pad-derived stromal [ 113]

cord mesenchymal stem

Chitosan Glycol chitosan/benzaldehyde Tunable mechanical properties; Good cell/material [ 117]

capped PEG interaction

biodegradability; Good biocompatibility

Field of Matrix Classiﬁcation Composition Main outcomes Refs.

Thiol-ene PEG Promotion of yaline-like engineered cartilage [ 119]

HA/PEG Outstanding load-bearing and shape recovery properties; [ 107b]

Short gelation time; Good cell viability, proliferation and

acid) poly(l-glutamic acid) mechanical properties

poly(l-glutamic acid) ectopic cartilage formation; Full injectability; Rapid in

polposus II/HA/Aggregan/microbial amount of sGAG; High conﬁned compressive strength

tetrasuccinimidyl cell/material interactions

gelatin/methacrylated HA of the integrin 𝛼v𝛽6-TGF-𝛽1 pathway

HA-pNIPAM Cell phenotype maintained; ECM generation promotion [ 127]

decrease in stiﬀness and modulus values of cellular

proliferation and chondrogenic diﬀerentiation of adult

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