Modular Microcarrier Technologies for Cell-based Bone Regeneration

Abstract

A variety of materials-based approaches to accelerate the regeneration of damaged bone have been developed to meet the important clinical need for improved bone fillers. This comprehensive review covers the materials and technologies used in modular microcarrier-based methods for delivery of progenitor cells in orthopaedic repair applications. It provides an overview of the field and the rationale for using microcarriers combined with osteoprogenitor cells for bone regeneration in particular. The general concepts and methods used in microcarrier-based cell culture and delivery are described, and methods for fabricating and characterizing microcarriers designed for specific indications are presented. A comprehensive review of the current literature on the use of microcarriers in bone regeneration is provided, with emphasis on key developments in the field and their impact. The studies reviewed are organized according to the broad classes of materials that are used for fabricating microcarriers, including polysaccharides, proteins and peptides, ceramics, and synthetic polymers. In addition, composite microcarriers that incorporate multiple material types or that are mineralized biomimetically are included. In each case, the fabrication, processing, characterization, and resulting function of the microcarriers is described, with an emphasis on their ability to support osteogenic differentiation of progenitor cells in vitro, and their effectiveness in healing bone defects in vivo. In addition, a summary of the current state of the field is provided, as are future perspectives on how microcarrier technologies may be enhanced to create improved cell-based therapies for bone regeneration.

Graphical Abstract

This review on osteogenic microcarriers outlines effective fabrication tools, material design properties, and applications for modular bone regenerative substrates.

Microcarriers in orthopaedic regenerative medicine

The need for new materials and methods to accelerate and improve bone healing outcomes has stimulated a broad field of research aimed at potentiating bone regeneration.^1,2 Although many bone fractures heal adequately without intervention, there is an unacceptably large number of cases each year in which bone heals sub-optimally, or in some cases fails to heal, which can lead to serious morbidity and chronic non-union.^3–5 In these cases, more advanced therapeutic approaches are needed, and there has been a strong emphasis on developing new, more potent bone fillers and engineered bone constructs that can regenerate bone even in challenging situations.³ The development of engineered bone has shown promise in creating bulk tissue constructs that can rapidly fill a defect and support surrounding tissue.² However, these approaches are hampered by the need for invasive surgery, a lack of new bone ingrowth, and potential compliance mismatch with surrounding tissues.^3,6

The drawbacks of monolithic, macroscale engineered constructs have motivated the search for alternative strategies to treating large bone defects. In particular, there have been increasing efforts to develop modular approaches, in which discrete biomaterial or engineered tissue units are combined to create bone fillers that can be handled as slurries or pastes.^6,7,8,9 The concept behind such semisolid systems is that these fillers can have transient fluidic properties sufficient for conformably filling irregularly-shaped cavities, while also allowing immobilization in the defect and providing support of the surrounding tissues as regeneration occurs. These bone-replacement materials are generally designed to be osteogenic in nature, such that they engender biomimetic mineralization of calcium phosphate minerals or allow the subsistence of cells that are capable of fulfilling this role. In addition, such modular systems can be designed for minimally invasive delivery, which has the potential to reduce residual pain, adverse scarring, and the risk of infection.

The field of orthopaedic repair has progressed as new biological approaches to tissue regeneration have been developed and proven effective. Over the past two decades, the subfield of orthobiologics has evolved to encompass a wide range of biological and bioactive materials that promote healing of musculoskeletal tissues.¹⁰ In addition to autografts and allografts, therapies involving growth factor delivery,^4,5,11 marrow-^4,10 and plasma-concentrates,^4,5,10 and transplantation of progenitor cells¹⁰ are being developed and tested in the clinic. These approaches require specialized biomaterials designs and chemistries to promote the desired bioactivity and resulting biological responses. In particular, the delivery of cells presents a challenge in ensuring the viability, maintenance, and stimulation of differentiated function that is required for an effective cell-based therapy. In response to these needs, a variety of cell-based approaches to orthobiologic bone repair have been developed, using a wide range of materials.¹³ In addition, a diverse set of cell types, sources, and formulations have been applied to potentiate the regeneration of bone.

The development of microcarriers for cell delivery combines the discrete modular approach to tissue engineering with the bioactive strategies of orthobiologics. The use of such engineered particulate materials is a promising strategy to create functional bone fillers. While there has been considerable effort to develop therapies based on macroscale particles with diameters in the millimeter range,^13,14 there has been less emphasis on establishing microscale approaches. Engineered microcarriers have the advantage that they can be designed to be conducive to cell attachment, to promote cell differentiation, and to degrade at a controlled rate. Their shape and size can be tailored to accommodate a range of cell densities and to form injectable microcarrier populations for downstream injectability. The composition and properties of such a carrier material can also be tailored to facilitate delivery, distribution, and immobilization of the microcarrier phase. These features make microcarrier-based delivery systems attractive for bone regeneration applications.

Microcarriers as vehicles for cell culture and delivery

Cellular therapies typically require large numbers of cells and the production of this biomass can be a barrier to creating an effective therapy. The high surface area to volume ratio afforded by the use of microcarrier-based cell culture, relative to standard two-dimensional culture in flasks or dishes, makes it attractive for generating large cell populations. A large surface area allows proliferation and expansion of cells adhered to the microcarrier surface. In some applications, biochemicals secreted by the adhered cells are harvested for therapeutic or other uses.^1,16 In other applications, the cells themselves are harvested by enzymatically releasing them from the microcarrier surface.³ In addition, the use of suspension culture techniques, typically in stirred bioreactors, allows efficient delivery of nutrients and controlled diffusion gradients. Such culture systems have been used for over 50 years for a variety of cell types of commercial interest in the biotechnology industry.¹⁶ However, microcarriers have not been as extensively used in the field of tissue engineering, despite their advantages in sustaining high volume cell growth.

Mesenchymal stromal cells (MSC) are adult progenitor cells that have been used widely in regenerative medicine. They can be harvested from a variety of tissues, including bone marrow and fat, and therefore offer a relatively accessible cell source for tissue engineering applications.^1,10 There is a broad literature on the isolation, expansion, characterization, and use of MSC for therapeutic purposes, though their exact mechanisms of action remain the subject of investigation. It is clear that these cells can secrete substances that potentiate regeneration through paracrine mechanisms,^11,12 and it has also been shown that they can have immunomodulatory effects.^17,18 The range of their differentiation capacity is still a subject of debate; however, it is generally accepted that MSC can act as progenitors for bone, cartilage, and adipose tissues.^1,19 In particular, it is well established that MSC form mineralized tissues when exposed to the appropriate environment, both in vitro and in vivo.^13,19 Therefore, MSC are a valuable cell source in bone regeneration applications, particularly in situations where native progenitor cells are depleted and/or cannot be mobilized to the site of injury. In these instances, transplantation of MSC offers a way to enhance bone tissue repair. However, there is a need for improved methods to culture, differentiate, and deliver MSC for therapeutic purposes.

The combination of MSC and microcarrier culture is a promising way to culture, differentiate, and deliver potent progenitor cells in large numbers. Careful selection and design of the materials used to fabricate the microcarrier substrate offers a mechanism for controlling cell proliferation and differentiation. Furthermore, the modular microcarrier format allows delivery of the cultured cells while still attached to a supportive and potentially bioactive substrate. The implanted cells and microcarriers can be designed to degrade at a controlled rate post-transplantation, thereby releasing the attached cells and allowing new tissue to replace the bone filling implant. Advances in biomaterials science have made it possible to control the properties of the microcarrier such that the desired biological functions are promoted over time. Therefore, microcarrier-mediated MSC delivery offers the possibility to tailor the spatial and temporal behavior of advanced bone graft materials.

The treatment of large and challenging bone defects using this strategy is particularly promising because these indications require a volumetric bone filler that has osteogenic properties. The microcarrier format allows large numbers of cells to be grown and differentiated under controlled conditions. It is likely that regeneration of larger defects will require the transplantation of billions and perhaps trillions of cells, and use of microcarriers in suspension culture and bioreactor systems facilitates scale-up and production of well-characterized batches for such therapeutic use. Furthermore, microcarrier-bound cells can be delivered directly to bone defects while leaving their cell-matrix contacts intact. The microcarrier matrix can therefore be designed to promote osteogenic differentiation, and this stimulus can be maintained both prior to and after cell transplantation. Importantly, the packed bed geometry of microcarrier populations facilitates diffusion and perfusion to supply nutrients to the cells post-transplantation, and therefore may perform better than other approaches in ischemic situations.

The following sections of this review provide a description of the materials and strategies that have been used to create microcarriers for cell-based regeneration of bone. It focuses on approaches designed for MSC and osteoblast culture and delivery, with an emphasis on the materials used to create microcarriers and their function in directing osteogenic processes. These materials are used alone and in combination to mimic key aspects of bone composition and function, while also providing a supportive and instructive substrate for living cells. In addition, this paper covers the fabrication and processing of microcarriers, and how they are cultured and delivered for bone regeneration applications. An impressive variety of approaches to modular microcarrier-enabled bone regeneration have been developed. However, these technologies have yet to have an impact in the clinic. This review is aimed at describing the current state of knowledge in this still growing field by summarizing the approach and main findings of the studies that have been done to date, and the impact that these findings may have on future studies and translation of microcarrier-based bone regeneration technologies to the clinic.

Microcarrier Fabrication, Processing, and Characterization

Many microcarrier types are produced by simple emulsification, which offers the advantages of bulk processing and relatively high throughput. In general, batch emulsification involves mixing two immiscible liquids to create a dispersion containing a separated colloidal phase within a bulk continuous phase. The mixing rate and properties of the colloidal and bulk phases can be varied to control the size and size distribution of the resulting particles, and emulsification has proven well-suited to creating polymer and polysaccharide particles on the nano- and micro-meter scale. Water-in-oil emulsion techniques are often used to suspend a polymer solution within a processing oil, creating a spherical discrete phase within the larger continuous, nonpolar phase. This discrete phase can be easily settled out of suspension and is often extracted with the aid of secondary interface-stabilizing surfactants.

Microfluidic flow-focusing droplet generation is a modified version of bulk emulsification aimed at tight control of particle size. In these microfluidic systems, an aqueous polymer solution and typically a nonpolar oil or other fluid are co-extruded in a specific geometrical arrangement to produce consistently-sized droplets.¹⁸ The size of these droplets can be altered through inputs of the junction geometry for the intersecting fluids and the flow rates of each fluid. However, these systems are limited in the viscosity of the polymer solutions that can be used, and microcarriers are necessarily created one at a time, which can prolong production times. One variation of the microfluidic flow-focusing technique uses a gas stream in the place of an oil phase. Such methods have been found particularly useful in the formation of ceramic particles.^8,21–26 Once particles have been formed and crosslinked, they are washed, dried, and sintered at elevated temperature.

Coacervation-based production methods for microcarriers are typically similar in general set-up to batch emulsification techniques (Fig. 1). The term, coacervation, is often used in reference to complex coacervation processes involving the mixture of two distinct solutions of a cationic macromolecular species and an anionic macromolecular species. This generally induces conglomeration and precipitation of a concentrated colloidal phase. Simple coacervation, which is more common to the process of microcarrier fabrication, refers to the phase separation of a macromolecular solution from a more dilute continuous phase to produce a relatively concentrated particulate colloidal phase. This process is often triggered by a change in temperature or pH, and has been used to create microcarriers for cell culture. Coacervate particles, like water-in-oil emulsified particles, can be collected by centrifugation and subsequently processed to stabilize and modify their morphology.²⁷

Fig. 1.

Schematic showing general microcarrier fabrication, processing, and application in bone tissue regeneration. Image contains altered elements from smart.servier.com.

Post-fabrication processing of microcarriers often includes crosslinking of the matrix to impart stability, reduce swelling, and resist dissolution. A variety of chemical, thermal and photonic crosslinking methods can be applied to microcarriers. For osteogenic applications, covalent crosslinking using glutaraldehyde or other aldehydes is often used to chemically bind primary amine groups between macromolecules.⁹ Carbodiimide-based crosslinking of amines using 1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) is also a commonly used stabilization method.²⁸ In specific instances, other crosslinkers may be of use, for example the use of divalent cations to stabilize alginate-based materials.²⁴ Similarly, formed and stabilized microcarriers can be characterized using a variety of standard techniques. The size and morphology of microcarriers can be measured using optical microscopy-based imaging software or laser diffractometry-based dimensional estimation equipment.^2,3,29 The composition of microcarriers can be determined using X-ray diffraction or Fourier transform infrared analysis.^2,6,30

Biomineralization is another common post-processing technique for microcarriers intended for bone regeneration applications.^31–35 Calcium mineral can be incorporated directly into the matrix of microcarriers to better simulate the composition of native bone.^2,3,29 In addition, incubation of microcarriers in high calcium concentration solution like SBF or Ringer’s solution can be used to induce the deposition of a mineral phase.^31–35 Control of the composition and thermodynamics of these techniques allows the tailoring of the mineral phase of microcarriers to promote desired functionality. Calcium phosphate mineral is often used because of its chemical similarity to carbonated apatite, a calcium-deficient form of hydroxyapatite that makes up as much as 65 wt% of adult bone.³⁶ Tricalcium phosphate is often added to microcarrier matrices,^37–39 and this mineral phase can be transformed into hydroxyapatite through preferential dissolution of less stable calcium phosphate minerals and their recrystallization into more stable forms, in a process governed by the Ostwald-Lussac rule of stages.⁴⁰ Calcium carbonate-based minerals can be used in a similar manner, and under controlled conditions can be transformed into calcium phosphate mineral phases that resemble those in biological hard tissue.^32,41 Furthermore, the inclusion of specific protein and polysaccharide materials in combination with such mineral phases is often applied to mimicking the physiological environment of bone.^2,3,6,29,42

Microcarrier Materials, Characteristics, and Function

Polysaccharide-composite microcarriers

Polysaccharide materials are generally abundant and well characterized, and they have been used extensively to produce microcarriers. Commercially available products such as the cellulose-based Cytopore¹⁶ and dextran-based Cytodex lines⁴³ are polysaccharide microcarriers with varying size, composition, and porosity. Although Cytopore beads are generally more suited to the culture of Chinese hamster ovary (CHO) epithelial cells, baby hamster kidney fibroblasts (BHK-21), HeLa cervix cancer epithelial cells, and Vero kidney epithelial cells, the collagen-coated Cytodex line has been used in a number of bone progenitor cell studies.⁴⁴ Cytodex beads made in the 150–250 μm range have been used in several studies to expand and osteogenically stimulate osteoblasts and osteoblast precursor cells for greater alkaline phosphatase (ALP) activity and mineral deposition.^45–48

Chitosan is a common polysaccharide biomaterial that has been tested as a base constituent for the construction of osteogenic microcarriers (Fig. 2). Porous microspheres of chitosan doped with up to 30% hydroxyapatite have been successfully prepared and characterized.⁵⁰ A relationship was established between the percentage of added hydroxyapatite and the microcarrier size. Pure chitosan microcarriers had diameters of 220–250 μm, which increased with hydroxyapatite addition to a diameter of 400–530 μm at 30% hydroxyapatite. This positive trend in size was accompanied by a negative trend in water sorption, which dropped from as much as 400% in pure chitosan to below 100% with mineral addition. Chitosan microcarriers have also been created using either coacervate precipitation or emulsion crosslinking, followed by immersion in simulated body fluid to create a coating of carbonated hydroxyapatite.²⁷ The biological effects of these chitosan-based modules (350–710 μm diameter) were evaluated using MC3T3-E1 pre-osteoblast cells. Cell proliferation was found to be markedly higher on the coacervate-precipitated carriers, relative to emulsion-crosslinked carriers, potentially due to the rougher surface topography of the former. Mineral coating further enhanced proliferation. Interestingly, mineral coating increased ALP activity of emulsion-crosslinked carriers, but did not have the same effect on coacervate-precipitated carriers, though the latter generally exhibited higher ALP activity. Collagen deposition increased after mineral coating in both microcarrier types, though uncoated coacervate-precipitated microcarriers showed more deposition than emulsion-crosslinked counterparts. These studies suggest that both surface chemistry and roughness, as well as the presence of a mineral phase can be used to enhance the osteogenic capacity of microcarriers.

Fig. 2.

Scanning electron micrograph of rat MSC 14 days after attachment to Cytodex 3 microcarriers. Image reproduced from Qiu et al.⁴⁹

Pullulan is a natural polysaccharide polymer that has been used widely in the food and pharmaceutical industries and is being investigated for medical applications.⁵¹ This material has been used to create porous microcarriers (150 μm average diameter), which were then seeded with SaOS-2 osteosarcoma cells and cultured in either static or dynamic suspension culture conditions.⁵² Pure pullulan microcarriers were compared to those with a mineral surface coating or coated with silk fibroin, as well to silica glass control microcarriers. Cell viability after seeding was generally below 50% for the pullulan-based carriers, and was only slightly higher on glass controls. Pure and silk-fibroin-coated microcarriers showed upregulation of ALP activity in both static and dynamic culture, while mineralized carriers showed the lowest activity, possibly due to the inhibitory effect that increased calcium ions may have on the ALP enzyme. Dynamic culture did not produce marked changes in ALP activity in pullulan-based carriers. However, there was a strong increase in ALP activity on glass microcarriers in dynamic culture, relative to static controls. This study suggested that pullulan-based carriers can support osteogenic differentiation, though response to dynamic culture was highest on very stiff silica substrates.

Protein- and peptide-composite microcarriers

Microcarriers created for use in osteogenic applications are very often composed of a collagen-derived base material (Fig. 3) supplemented with calcium-based compounds to further enhance functionality. These two components roughly mimic the composition of the native bone extracellular matrix, which is essentially a mineralized matrix of collagen.³⁶ Gelatin is a mixture of peptides derived from the hydrolysis of collagen, and is therefore often used as a microcarrier base material. Collagen and collagen peptides have the advantage that cells can recognize, bind to, and degrade these materials, giving them enhanced biological functionality relative to most synthetic materials. These proteins and similar protein-based materials can be extracted from animal tissues using a variety of techniques, which impart specific physical and biological properties on the resulting matrix. Similarly, there are a variety of forms of calcium phosphate compounds that have been used to augment protein-based microcarriers. The selection of base material and filler can be leveraged to control cell function, including for osteoconductive, osteoinductive, and osteogenic purposes.

Fig. 3.

Cultispher-S gelatin microcarriers seeded with osteogenic cells. Panels (a) and (b) are stained with propidium iodide to show cell viability at day 14. Panel (c) shows hematoxylin & eosin staining at day 14. Panel (d) shows Masson’s Trichrome staining at day 28 of culture. Reprinted (adapted) with permission from Declerq et al.⁹

Gelatin-based microcarriers are available commercially. Cultispher-S microcarriers are composed of highly crosslinked porcine gelatin,^9,19 and are designed with high porosity for cell loading. It was found that rat MSC exhibited markedly higher levels of the osteogenic markers osteocalcin and alkaline phosphatase when cultured on Cultispher-S microcarriers, compared to conventional tissue culture polystyrene.⁵³ When tested in vivo, it was shown that MSC cultured on Cultispher-S carriers resulted in improved trabecular bone formation relative to cell-free microcarriers in long bone defects,⁵³ and similar effects were observed upon application to periodontal defects.⁵⁴ Earlier studies also investigated microcarriers composed principally of collagen Type I (Cellagen™) for the expansion of human osteoblasts. It was demonstrated that osteoblasts cultured on collagen microcarriers in spinner flask culture displayed a higher proliferative capacity over a 15-day period, when compared to cells cultured on tissue culture polystyrene. Spinner culture on collagen microsphere substrates was also shown to dramatically upregulate osteocalcin levels relative to monolayer cultures.⁴³

In experimental systems, gelatin-based microcarriers have been augmented with various forms of calcium phosphate mineral. Gelatin mixed with tricalcium phosphate (TCP) was used to create microcarriers by emulsification in olive oil,²⁸ followed by crosslinking using 1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS) chemistry. Subsequently, the microcarriers were incubated in Ringer’s solution for seven days to convert TCP to calcium-deficient hydroxyapatite via hydrolysis, as in a preliminary exploratory study.³⁷ The resulting microcarriers (100–300 μm diameter) were seeded with SaOS-2 osteosarcoma cells and were used in static and dynamic suspension culture studies, with Cytodex-3 microcarriers serving as controls. In static conditions, cells attached to the gelatin-HA microcarriers but no statistical increase in cell number was observed over three days of culture, while cell number on Cytodex-3 microcarriers increased approximately 4-fold over the same time period. In contrast, under dynamic culture conditions, cell proliferation was clearly enhanced on gelatin-HA microcarriers, compared to Cytodex controls. This study illustrates that interactions between the cell substrate and the culture conditions that can lead to differences in cell behavior, and in particular highlights the benefit of using gelatin-based microcarriers in dynamic suspension culture.

Other studies have examined the mode of incorporation of calcium phosphate minerals into gelatin-based materials. Microcarriers formed of apatite and gelatin were fabricated through a sol-gel process by mixing gelatin with calcium hydroxide and subsequent reaction with phosphoric acid.² The resulting apatite-gelatin microspheres (112 μm average diameter) were crosslinked using EDC chemistry. The sol-based emulsification method was found to impart a finer, nanoscale distribution of the calcium orthophosphate apatite crystal phase within the continuous gelatin phase, when compared to microcarriers made by direct mixing of pre-formed apatite powder into gelatin solution. Culture of MG-63 human osteosarcoma cells on the sol-formed microcarriers showed that they supported cell proliferation and osteogenic function. In a related study, apatite mineral was formed inside glutaraldehyde-crosslinked gelatin nanoparticles (230–250 nm diameter) created using a “miniemulsion” technique.⁶ This method of biomimetic mineralization promoted the constrained formation of a single crystalline hydroxyapatite phase throughout the gelatin spheres. These gelatin-CaP particles were too small to support the culture of mammalian cells, though the same technique could potentially be used for larger particles. These studies are important because they demonstrate control of the micro- and nano-scale structure of mineral in gelatin materials, which can be tailored to mimic native bone mineral.

Gelatin-based microcarriers have also been used in other formats aimed at regenerating bone. Addition of gelatin microspheres (20–100 μm diameter range) to MSC cultures in attachment-inhibiting cultureware demonstrated that aggregates of cells and microspheres formed over time.⁴² The presence of microspheres in the aggregates resulted in higher cell viability and production of alkaline phosphatase, a marker of osteogenic differentiation. It was proposed that the inclusion of gelatin particles increased the porosity of the aggregates, and therefore enhanced nutrient and oxygen delivery. In a separate study, direct precipitation of a mixed solution of gelatin and calcium phosphate was used to fabricate small (<10 μm diameter), spherical, and highly porous particles that were conducive to attachment, viability, and proliferation of human osteoblast-like G-292 cells.¹⁴ Implantation of composites of these microspheres and cells in a critical size rat calvarial defect model resulted in strongly improved bone regeneration at four and eight weeks, relative to clinically-used fibrin glue and a commercially available bone filler material (Osteoset® calcium sulfate mini-beads). In another study, gelatin microspheres (50–100 μm diameter) were incorporated into injectable co-N-isopropylacrylamide macromer materials designed for in-situ gelling.⁵⁵ The gelatin phase acted as a substrate for cell attachment and as an enzymatically-degradable porogen. Testing in a rat critical size cranial defect model revealed a greater level of mineralization and bony bridging in microsphere-containing samples, relative to controls. These studies highlight the potential improvement in bone regeneration achieved when combining osteogenic cells and a rationally designed carrier material, relative to current clinical options.

Other forms of collagen peptides and preparations have also been combined with calcium mineral to form microcarriers. Hydroxyapatite particles dispersed in reconstituted fibrous collagen Type I were prepared by emulsification in olive oil and crosslinked with glutaraldehyde.³ Increased stirring speed during emulsification resulted in a decrease in average microcarrier diameter, which ranged from 220–1040 μm. A further decrease in microcarrier diameter could be achieved by the addition of a surfactant during emulsification, down to a range of 75–300 μm average diameter. Primary rat osteoblast cells proliferated and increased their production of alkaline phosphatase when cultured on these microcarriers. In a follow-on study, the ultrastructure of the matrix in similar microcarriers was examined.²⁹ The hydroxyapatite was distributed evenly in the matrix and did not hinder collagen fibril formation. Primary rat osteoblasts cultured on these microcarriers under osteogenic conditions proliferated and maintained metabolic activity, and also mineralized the underlying collagen-HA matrix. In a third study, similar microcarriers were made to have bone-mimetic composition of 35:65 wt% collagen:hydroxyapatite at an average diameter of 500 μm.⁵⁶ The collagen-HA microcarriers were compressed into discs and were press-fit into a critical size cranial defect in the rat, without the addition of exogenous cells. After 16 weeks if implantation, the collagen-HA microcarriers were completely resorbed and showed good bone ingrowth, whereas pure hydroxyapatite control implants were not completely resorbed.

Collagen microcarriers incorporating tricalcium phosphate (TCP) and calcium phosphate cements are of interest because of their similarity to bone matrix and ability to support osteoblast function.³⁹ Microcarriers made from CPC with a range of particle sizes became more spherical and cell-adhesive when collagen was added to the matrix.³⁸ It was shown that nanoscale calcium phosphate cement particles (2.4 nm average diameter) had the effect of increasing ALP activity when using Saos-2 osteosarcoma cells, whereas cell proliferation decreased on microcarriers relative to on silica glass controls. In a follow-on study, similarly-formulated composite collagen and calcium-deficient hydroxyapatite microcarriers were implanted in femoral defects in a rabbit model.⁵⁷ Compared to injectable calcium phosphate cement, both with and without the addition of collagen, the microcarriers produced a ten-fold increase in bone ingrowth at 3 months. These results again emphasize that the geometry and composition of microcarriers can be used to tailor their function in bone regeneration applications.

Sol-gel preparation can also be used to create apatite-loaded collagen microcarriers, in a process analogous to that developed for gelatin.² Pure collagen and collagen-apatite microcarriers (166 μm average diameter) were made using peptides derived from collagen sponge material using emulsification and subsequent crosslinking with EDC-NHS chemistry.⁵⁸ Apatite was then precipitated within the collagen microcarrier matrix as homogeneously-dispersed nanocrystals, to a loading of approximately 30 wt%. Pure collagen microcarriers were somewhat smaller (120 μm average diameter), again reflecting a relationship between mineral loading and particle size. Rat bone marrow-derived MSC cultured on these collagen-apatite microcarriers proliferated more rapidly compared to those on pure collagen microcarriers. In addition, MSC grown on collagen-apatite microcarriers expressed higher levels of the osteogenic marker ALP, relative to MSC cultured on pure collagen microcarriers or on standard tissue culture plates. These findings reinforce the use of a defined mineral phase in enhancing the function of peptide- and protein-based microcarriers.

Protein-based microcarriers designed to fully envelop progenitor cells for delivery in bone regeneration applications have also been developed. Composites of collagen Type I and the polysaccharide agarose were used to prepare microbeads (30–150 μm diameter range) by emulsification, with human MSC embedded directly within the microbead matrix.⁵⁹ 3D microcarrier culture resulted in increased bone sialoprotein production and calcium deposition, relative to 2D controls. Similar microbeads (90–290 μm diameter range) were also fabricated using a collagen-chitosan matrix with embedded human MSC (Fig. 4).⁶⁰ High cell viability was supported in these microcarriers and osteogenic capacity was demonstrated. Populations of microbeads could be concentrated into a paste for delivery through a standard 25G needle without loss of cell viability. Implantation of collagen-chitosan microbeads containing bone marrow mononuclear cells supplemented with purified MSC in an ectopic site in the rat resulted in markedly enhanced bone formation, relative to controls.⁶¹ An extension of this work involved augmenting the collagen-chitosan matrix with exogenous hydroxyapatite, and examining bone regeneration in an orthotopic, critical size cranial defect model in the mouse.⁶² This study showed the value of pre-differentiating MSC prior to implantation, and showed that such microbeads could be used to conformally fill and heal a critical size defect.

Fig. 4.

Viability staining of MSC embedded in modular microbeads with varying chitosan/collagen ratios at days 1 and 9 in culture (scale bar represents 200 μm). Cytoplasm of living cells is stained green and the nucleus of dead cells is stained red. Images reproduced with permission from S. Karger AG, Basel, Wang et al.⁶⁰

Silk fibroin has been investigated in a variety of regenerative medicine applications,^18,20,52 including for the production of osteogenic microcarriers. Gelatin-fibroin microcarriers were prepared at a 30:70 wt% gelatin:fibroin composition, both with and without mineralization using calcium phosphate.¹⁸ MSC attached and spread on these microcarriers and proliferated more robustly on gelatin-fibroin substrates than on Cytodex-3 controls, but it was observed that mineralization greatly decreased cell proliferation. In contrast, ALP activity was strongly upregulated on gelatin-fibroin microcarriers that had been mineralized with calcium phosphate. The effect of composition on the function of gelatin-fibroin was further examined using microcarriers (300–400 μm diameter range).²⁰ The seeding efficiency and subsequent proliferation of rat MSC seeded on these microcarriers decreased with increasing silk fibroin content. However, a 75:25 wt% fibroin:gelatin composition was found to be the most osteogenic in terms of osteopontin expression, relative to control Cultispher-S microcarriers. These studies suggest that mineralization of non-collagenous matrices is also beneficial in achieving osteogenic differentiation of progenitor cells.

Ceramic and ceramic-composite microcarriers

Bioactive glasses are a class of calcium sodium phosphosilicate ceramics that have been used widely in bone healing applications.^5,11,63 Their use as microcarriers typically involves pre-coating or otherwise incorporating a calcium phosphate mineral phase. Optimization of the microcarrier coating procedure has shown that extended sequential exposure to Tris-HCL followed by tissue culture medium results in a more functional coating, with a transition over time from amorphous calcium phosphate to carbonated hydroxyapatite, and a resulting positive effect on the osteogenic differentiation of MSC.⁶⁴ It has also been suggested that carbonated hydroxyapatite coatings preferentially adsorb fibronectin and other cell-binding proteins, which may enhance biological function.^7,65 Microspheres (100–150 μm diameter) created using bioactive glass have been studied for orthopaedic use as solid microparticles, hollow microspheres, and as part of polymer composite microspheres that were coated with mineral through submersion in simulated body fluid.^66–67 Cell culture studies showed that bone marrow stromal cells attached to the microcarriers and produced extracellular matrix that could be mineralized. This process also produced larger cell-microcarrier aggregates. Similarly mineralized bioactive glass microshells were shown to support osteogenic differentiation of rat MSC, as indicated by alkaline phosphatase, collagen type I, and osteopontin expression.⁴⁹ Mineral deposition by rat MSC was also demonstrated on such microspheres using scanning electron microscopy.^13,69 Composite microspheres of bioactive glass and poly(lactic-co-caprolactone) incubated in simulated body fluid (SBF) for one week exhibited complete CaP mineral coverage along with inducing significantly higher ALP activity than purely poly(lactic-co-caprolactone) microspheres on day 14 and 21 of culture in osteogenic media.³⁵ Other work on phosphate-based bioactive glass microspheres has suggested that the release of ions is important to their stimulation of bone regeneration, as well as the need to allow for appropriate degradation rates to support collagen deposition and matrix remodelling.^11,63,70

Calcium titanium phosphate (CTP) microcarriers are a material variation on bioactive glasses.^8,21–23 Particles of CTP (600 μm average diameter) were compared to polystyrene microcarriers (200 μm average diameter) in terms of the support and differentiation of rat MSC.⁸ Attachment efficiency of MSC to CTP microspheres was lower than that on corresponding polystyrene microcarriers, though the cells proliferated over two weeks in culture on both substrates. However, ALP and osteocalcin secretion was shown to be higher on CTP microcarriers, relative to polystyrene controls. Another formulation of composite titanium phosphate glass microcarriers (50–100 μm diameter range) has also been compared to similarly-sized silica glass microspheres.^71–72 These microcarriers supported the proliferation of MG-63 osteosarcoma cells in static and spinner flask culture over one week. In a similar study using human MSC, it was observed that CTP microcarriers potentiated BMP expression, relative to silica glass, and in particular that osteopontin was highly upregulated over time on CTP microcarriers. These studies suggest that the chemical structure of calcium titanium phosphate microcarriers is conducive to the osteogenic differentiation of bone progenitor cells.

Hydroxyapatite is a ceramic of particular interest in orthopaedic regeneration applications because it closely mimics the mineral phase of native bone.^2,11,36 This mineral has been incorporated as an additive in microcarriers based on a variety of materials, and has also been used as the primary constituent in microparticles designed for cell delivery. Hydroxyapatite microspheres (400–550 μm diameter range) were shown to support the adhesion, proliferation, and osteoblastic lineage of human MG-63 osteosarcoma cells (Fig. 5).^22,26 The effect of the density and porosity of hydroxyapatite particles was examined using microcarriers (200–700 μm diameter range) designed for the delivery of goat MSC.⁷³ Larger, microporous, MSC-seeded carriers resulted in no visible bone formation when implanted subcutaneously in athymic mice, and produced only a vascularized fibrous tissue. Smaller, dense microcarriers exhibited higher cell attachment efficiency, and resulted in trablecular-like bone formation in a subcutaneous site. The effect of surface topography on cell seeding was examined using spherical, hollow hydroxyapatite microcarriers (360 μm average diameter) seeded with murine osteoblasts.⁷⁴ These microcarriers supported cell attachment and proliferation both on the outer concave and inner convex surfaces. These findings emphasize the importance of microcarrier morphology and topography on the response of seeded progenitor cells.

Fig. 5.

Upper panels: scanning electron micrographs (left: x110; right: x1000) of osteogenic cells on hydroxyapatite microcarriers after day 3 of culture. Lower panels: confocal laser scanning micrographs of osteogenic cells on hydroxyapatite microcarriers (x100) on day 5 of culture. Reprinted from Mateus et al.²⁶ with permission from Elsevier.

Tricalcium phosphate (TCP) is a ceramic with similar composition to hydroxyapatite, but which typically has a lower Ca:P ratio than native hydroxyapatite. TCP can be converted to hydroxyapatite by sintering and chemical processing,^{28,31,37–39,57} and the latter is generally found to be more osteoconductive.⁷⁵ Microcarriers (100–250 μm diameter range) have been formed out of composites of HA and TCP and were seeded with bone marrow-derived MSC.⁷⁶ Implantation of MSC-seeded HA-TCP microcarriers into both calvarial and mandibular defects in the mouse resulted in significant new bone formation by six weeks. In contrast, unseeded HA-TCP microparticles implanted into similar defect sites resulted in poor bone formation even at very long time points. Perforated HA-TCP microcarriers (370 μm average diameter) were also prepared and used as a culture substrate for human adipose-derived MSC.^77–78 Calcium deposition increased on these microcarriers over time in response to osteogenic stimulation in the culture medium, although the response was not as strong as from cells grown on tissue culture plastic. Implantation of unseeded versions of these HA-TCP microcarriers in rabbit calvarial defects resulted in the production of mature bone within the microcarrier cavities and lamellar osteons over six weeks. These observations suggest that in these composite microcarriers the HA component nucleates mineralization, while the degradation of the TCP phase provides space for new tissue formation.

Ceramic materials have also been augmented with other components to enhance the bone regeneration capability of microcarriers. Strontium-doped hydroxyapatite microspheres (520 μm average diameter) were combined with an injectable, in-situ crosslinkable RGD-alginate carrier gel to treat critical size femoral defects in the rat.²⁴ Micro-computed tomography and histological analysis indicated more robust bone formation in strontium-augmented implants, relative to strontium-free controls. Microcarriers augmented with strontium also degraded more quickly, leaving less residual material in the implant site. The result was increased collagen deposition and new bone formation in the interstitial space between microcarriers. Composite TCP-alginate microcarriers (100–500 μm diameter range) were fabricated and increasing TCP content was correlated with an increase in diameter.³¹ These carriers were subsequently incubated in SBF and their pore size could be modulated through control of the freezing temperature during lyophilization, with increasing freezing temperature resulting in larger pores. Using MC3T3-E1 pre-osteoblast cells, it was shown that cell proliferation and ALP activity under osteogenic conditions were similar on TCP-alginate microcarriers, relative to tissue culture plastic controls, suggesting that such microcarriers support the osteoblastic differentiation of progenitor cells.

Synthetic Polymer-composite Microcarriers

Polystyrene is widely used in tissue cultureware because it can be plasma treated to promote cell attachment.⁷⁹ This polymer can also be used to fabricate spherical microparticles, which have found broad utility in the biotechnology field as microcarriers.^80–81 Uncoated and collagen-coated SoloHill® polystyrene microcarriers (125–210 μm diameter range) were seeded with human MSC derived from bone marrow, placenta, or embryonic stem cells.¹⁷ Similarly, untreated or collagen-coated polystyrene tissue culture plates served as controls. It was observed that MSC cultured on collagen-coated microcarriers exhibited markedly higher ALP activity, collagen secretion, and calcium deposition compared to culture plate controls, even in the absence of osteogenic stimulation by growth factors. Both disruption of cytoskeletal actin using latrunculin B and inhibition of actomyosin contraction using blebbistatin reduced the osteogenic response, suggesting that in this system the microcarriers induced osteogenesis in MSC through enhancing cytoskeletal tension.

Polylactic acid (PLA) has been used widely for regenerative medicine applications, including for orthopaedic repair (Fig. 6).^32–34 Microspheres of PLA used for bone regeneration generally incorporate a ceramic phase to enhance osteogenic properties. The addition of calcium phosphate mineral can also mediate the production of acidic degradation products of PLA, and can act as a template for further biomineralization. Hollow spheres (500–1000 μm diameter range) were fabricated from a composite of PLA and calcium carbonate (CaCO₃) via emulsion.³² Subsequent immersion in SBF for one week resulted in production of a carbonated hydroxyapatite layer, as demonstrated by x-ray diffraction analysis. Similar porous PLA microspheres (110–250 μm diameter) were produced and treated in sodium hydroxide solution to hydrolyze surface functional groups.^33–34 Subsequent mineralization studies in SBF showed that pre-hydrolized microcarriers mineralized to a greater degree than untreated controls, and that the degree of mineralization increase with time after day 5. Seeding of human osteoblast-like MG-63 cells on mineralized microcarriers showed high cell viability (>80%) over five days of culture.

Fig. 6.

Scanning electron micrographs of MG-63 cells on day 3 of culture on (a, b) PLA microcarriers, (c, d) 8-day mineralized PLA microspheres, and (e, f) 17-day mineralized PLA microspheres. Reprinted from Shi et al.³³ with permission from Elsevier.

Composite poly(lactide-co-glycolide) (PLGA) microcarriers made with 50 wt% hydroxyapatite exhibited increased attachment efficiency of mouse OCT-1 osteoblast-like cells with increasing sodium hydroxide treatment.⁸² Cells proliferated over time in culture and alkaline-treated microcarriers were shown to be more osteogenic than untreated controls. PLGA microcarriers have also been used in dynamic culture systems for SaOS-2 human osteosarcoma cells.⁸³ While rotating dynamic culture decreased the proliferation rate, it also increased ALP activity in this cell line. Surface-mineralization of PLGA microcarriers also increased the attachment efficiency of rat osteoblast cells, relative to untreated controls.⁸⁴ Similarly, surface mineralization was shown to increase bone regeneration by these cell-seeded PLGA microcarriers when implanted into subcutaneous sites in athymic mice for 6 weeks. PLGA-based microcarriers have additionally been shown to generate nearly complete bone restoration in rat critical-size calvarial defects after 12 weeks of implantation.⁴¹

Summary and future perspectives

A wide variety of microcarriers have been developed with the target of augmenting bone regeneration. The studies reviewed in this paper are characterized by the rational combination of materials and chemistries to achieve desired physical and biological functions. Polysaccharides, proteins, peptides, ceramics, and polymers all have applied in this way. Composites of these materials are often used in an effort to harness and combine the desired properties of the individual components to create a more functional matrix. In some cases, these materials directly mimic the composition of biological bone tissue. In other cases, they are designed to promote cell attachment and function for the purpose of potentiating the biological response. Direct addition of calcium phosphate compounds or promotion of a biomineralization is a key strategy in creating many of these microcarrier types, since this mineral phase is a major component of bone tissue and has been demonstrated to have osteogenic effects on progenitor cells. Overall, there has been a remarkable diversity in the approaches taken to creating microcarriers for orthopaedic applications, and to designing their composition for enhanced biological function.

The use of cell-based approaches to orthopaedic tissue regeneration is more complex than purely materials-based approaches. However, only cells can produce new bone and therefore if appropriate cell types are not available at the site of injury, they must either be recruited endogenously or delivered exogenously. In large and ischemic bone defects, recruitment of progenitor cells is impaired, and cell delivery has the potential to greatly augment the healing process. Microcarrier-based strategies to cell therapy have the advantage that cells are delivered on a substrate that can be designed to direct their differentiation and function. In addition, the microcarrier material acts as a space-filling extracellular matrix that can have biological and mechanical function. Importantly, populations of microcarriers can often be delivered as a moldable paste or putty, and therefore can conformally fill defects. In most cases, these microcarrier-based approaches do not provide load-bearing mechanical stability. However, cell-seeded biomaterial microcarriers offer new and potentially improved options to fill bone defects that may be superior to current approaches in healing challenging cases.

The literature reviewed in this paper emphasizes the materials used to create microcarriers, and how the properties of the materials affect cell function. Materials chemistry is an important component of cell-matrix interactions, since cells bind directly to the microcarrier substrate and receive both physical and biological signals via that binding. Similarly, the topography of the substrate can be an important determinant of cell function, particularly in the case of progenitor cells such as MSC. Surface roughness, geometry, and porosity all have been demonstrated to affect cell differentiation, and these features can be designed into microcarriers in a variety of ways. Similarly, mechanical stiffness of materials is known to be a strong determinant of progenitor cell fate, particularly in orthopaedic applications. In addition, microcarrier materials can be designed to be more or less resistant to degradation in the physiological environment, which enables control over the dynamics of resorption and replacement of microcarriers by new biological tissue. Taken together, there are a wide range of options of materials design parameters that can be employed to make microcarriers optimally functional and effective in bone regeneration applications.

While remarkable progress has been made in the design, fabrication, characterization, and application of microcarriers for orthopaedic applications, there are exciting opportunities for further improvement of these technologies. New materials and composites are being developed that may facilitate tailoring of the cell-instructive properties of microcarriers. In particular, a better understanding and tighter control over material mechanical properties may allow more targeted cell differentiation. Materials can also be designed to change their properties dynamically over time or upon application of a specific stimulus, so that integration into the host is enhanced. Immobilization and release of bioactive factors is an area that has also been extensively studied in regenerative medicine, but has not been applied widely for microcarrier-based applications. Growth factors, function-specific ligands, gene delivery vectors, and a variety of other stimuli can be incorporated into and presented by the microcarrier matrix. Genetic modification of cells to be better suited for microcarrier delivery, and to be more effective upon delivery, is another exciting avenue that has not been extensively pursued in the field of microcarrier technology. Finally, there is a growing recognition that control of the biological response, and in particular the inflammatory and immunological response, is a key to achieving functional tissue regeneration. There remains great potential in using rationally-designed microcarriers for the delivery and directed differentiation of progenitor cells, with the goal of rapidly and effectively regenerating bone tissue.

Table 1.

Characteristics and applications of osteogenic microcarriers.

Microcarrier bulk materials	Diameter (μm)	Bone progenitor cells	Target (in-vitro/in-vivo)
Dextran (Cytodex-1,2,3) and gelatin (Cytodex-2,3)	150–250	Osteoblasts, osteoblast precursor cells, MC3T3-E1 cells, fetal rat calvarial cells, rat bone MSC	In-vitro static and dynamic (high aspect ratio vessel) culture
Chitosan and hydroxyapatite	220–710	MC3T3-E1 cells	In-vitro static culture
Pullulan and silk-fibroin	100–215	Human osteosarcoma SaOS-2 cells	In-vitro static and dynamic (orbital shaker) culture
Gelatin (Cultispher-S, etc.) and α-tricalcium phosphate, hydroxyapatite, or silk-fibroin	20–652.3	UMR-106 rat osteosarcoma cells, rat bone MSC, rat ear MSC, SaOS-2 cells, human osteosarcoma MG-63 cells, mouse bone MSC, MC3T3-E1 cells	In-vitro static, poly(lactide-co-ε-caprolactone) scaffold, and dynamic (spinner flask) culture; In-vivo rat subcutaneous site, long-bone defect, and periodontal defect
Type I Collagen (Cellagen®, etc.) and hydroxyapatite, α-tricalcium phosphate, calcium-deficient hydroxyapatite, apatite	30–1038	Human and rat osteoblasts, SaOS-2 cells, rat bone MSC, human MSC	In-vitro dynamic (spinner flask) and static culture; In-vivo rat calvarial defect and rabbit femoral defect
Calcium silicate	241	MC3T3-E1 cells	In-vitro static culture
45S5 Bioglass and polylactic acid or poly(lactic-co-caprolactone)	100–432	Rat bone MSC, MC3T3-E1 cells	In-vitro dynamic (rotating-wall or high aspect ratio vessel) and static culture
Phosphate-based (0.4P2O5:0.16CaO:0.175–0.2Na2O:0.24MgO:0–0.025Ti) glass	100–200	Autologous bone marrow concentrate cells	In-vivo sheep knee bone defect
Calcium titanium phosphate and alginate	50–796	Rat bone MSC, MG-63 cells	In-vitro static and dynamic (spinner flask) culture
Hydroxyapatite and alginate, α-tricalcium phosphate, or β-tricalcium phosphate	100–749	MG-63 cells, goat MSC, MC3T3-E1 cells, human osteoblastic cells, human bone and adipose MSC	In-vitro static culture; In-vivo mouse subcutaneous site, mouse calvarial defect, mouse mandibular defect, rabbit calvarial defect
α-Tricalcium phosphate and alginate	100–500	MC3T3-E1 cells	In-vitro static culture
Polystyrene and type I collagen	125–212	Human placental multipotent cells, human embryonic stem cell-derived MSC, bone MSC, mouse MSC (C3H10T1/2)	In-vitro static culture
Polylactic acid	150–250	MG-63 cells	In-vitro static culture
Poly(lactide-co-glycolide) and hydroxyapatite, calcium carbonate, or polyphosphate	50–860	Mouse osteoblast-like OCT-1 cells, SaOS-2 cells, rat osteoblasts, human MSC	In-vitro static and dynamic (high aspect ratio vessel) culture; In-vivo mouse subcutaneous site and rat calvarial defect

Abbreviations

ALP

Alkaline Phosphatase

BMP

Bone Morphogenetic Protein

CPC

Calcium Phosphate Cement

CTP

Calcium Titanium Phosphate

EDC

1-Ethyl-3-(dimethylaminopropyl) carbodiimide

HA

Hydroxyapatite

PLA

Polylactic Acid

PLGA

Poly(lactic-co-glycolic)

SBF

Simulated Body Fluid

TCP

Tricalcium Phosphate

Cell line abbreviations:

G-292

Human Osteosarcoma Cell Line

MC3T3-E1

Mouse Calvarial Osteoblast Precursor Cell Line

MSC

Mesenchymal Stem Cells

MG-63

Human Osteosarcoma Cell Line

OCT-1

Osteoblast-like Cells

SaOS-2

Human Osteosarcoma Cell Line