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Bone and cartilage injuries deriving from trauma, tumors, inflammation diseases, as well as natural aging, cause debilitation among affected individuals and represent a challenge for medicine. In particular, bone is subjected to frequent age and disease-related degeneration with mass decrease: the osteoporosis. Moreover tumors, trauma and chronic inflammation can determine localized bone loss. On the other hands, a major cause of disability in middle-aged and older people is represented by joint pain. Thus cartilage degeneration due to primary osteoarthritis, trauma and injuries resulting from sport activities are all possible causes of this kind of pain [1]. As the cartilage is a tissue with a little self-regenerative capacity, any alteration of its integrity might be carried on for years and eventually lead to further degeneration [2].

Frequently severe joint pain appears to be debilitating and this complication has motivated the research for scientists and surgeons to find a way to repair or regenerate lost cartilage. Given that it is a tissue with very distinctive properties, little success has been obtained until now.

Differently from bone, which has a consistent number of osteoblast precursors in the periosteum and bone marrow, cartilage has a matrix with no vascularization, with a consequent reduction of the tissue ability to recruit endogenous chondroprogenitor cells for healing; it is a flexible elastic tissue, compression resistant, it is able to distribute the loads to which it is subjected. It makes difficult the cartilage replacement with a tissue or any other designed device [3, 4].

Both in bone and in cartilage lesions it is important to distinguish between repair and regeneration. While a wound repair, which is mainly an inflammatory process with the subsequent recruitment of cells able to modulate the reparative processes occurs easily [5], the regeneration of the injured tissue consisting in an architectural and functional recovery hardly develop especially in cartilage. This process is thought to be due to the activation of resident stem cells or to the proliferation of quiescent cells with the restitution ad integrum of the tissues. In this case Mesenchymal Stem Cells (MSCs) are necessary [6, 7].

The current clinical practice for hard tissues defects repair employs autologous and allogeneic grafts. These strategies provide a scaffold support and a source of cells for the new tissue formation, but both approaches are often associated with post surgery complications as donor-site morbidity, pain, non-union fractures and infections [8, 9]. Therefore, the reconstruction of bone and cartilage defects is a challenge for regenerative medicine because of inadequate current treatments. To this purpose alternative approaches are desirable to address other possible strategies for hard tissues regeneration. Tissue engineering, relying on cell biology and material chemistry knowledge, aims to achieve the development of tridimensional substrates that can substitute bone implants. This approach can be optimized with the use of stem cells in particular Mesenchymal Stem Cells (MSCs), based on their direct ability to generate progenitors able to differentiate in chondroblasts and osteoblasts. In vitro differentiation of MSCs, represents the initial step to achieve the tissue regeneration, but to repair critical size defects, the cells must be seed on tridimensional scaffolds in order to recreate the structure and functionality of the lost tissues. Furthermore the integration of the new bone and cartilage with the resident tissue is needed, together with the appropriate degradation of the scaffold at the end of the healing process [10]. MSCs are stem cells of mesodermal origin and are responsible for the differentiation of properly defined connective tissues: the specialized ones, muscle tissues and also some epithelial tissues. MSCs, obviously forming the embryo and fetal mesenchyme, are present in many adult organism sites. Thus MSCs have the role to develop diverse differentiated tissues during embryogenesis and allow tissue renewal and repair during all life. MSCs residence in adults has been found in several locations and is still under investigation but bone marrow has been identified as the main residence site, here they are also defined as bone marrow stromal cells. Many other sites such as dental tissues, adipose tissue and peripheral blood has been demonstrated to contain a population of MSCs [11]. The research in the field of tissue regeneration has reached several goals in early clinical trials of therapies based on living cells. However, methods to regenerate bone and cartilage using living autologous cells are still under investigation. Thus, autologous bone still remains the gold standard for the repair of damaged bone tissues. Cartilage regeneration is still a step behind due to the lower chondrocyte proliferation and to the minor availability of convenient harvesting sites. Both bone and cartilage regeneration due to the morphology of the tissues is based on cell integration with opportune scaffolds. The choice of the most appropriate scaffold would bring optimal biological, chemical and geometrical characteristic to generate the more efficacious microenvironment to support the new bone formation. As first, the geometry of the scaffolds must be designed to ensure the correct transport of gas, nutrients and metabolites to the cells, but providing on the same time the adequate load support during the bone reconstruction therapy [12]. An adequate internal porosity in 3D scaffolds will better mimic the natural structure and the mechanical properties of bone tissue [13]. Furthermore, the material composition and surface properties of the scaffolds will help MSCs adhesion and migration, thus favoring the osteogenic and chondrogenic differentiation. Since mineral bone matrix is naturally composed of calcium in hydroxyapatite (HA) crystals, to obtain bone regeneration, bioceramics able to incorporate hydroxyapatite or tricalcium phosphates (TCP), have been fabricated to mimic the tissue composition and stimulate the osteogenic differentiation of MSCs [14, 15]. These type of bioceramics have been also shown to facilitate the integration of the scaffold following in vivo implantation [16]. Synthetic polymers as polycaprolactone and polyethylene glycol have been used to generate scaffolds with the recent technology of 3D printing. These materials allow incorporating peptides containing the cell-binding sequence arginine-glycine-aspartate (RGD) or load regulatory molecules (BMP-2) to be gradually released, thus enhancing osteogenic differentiation of MSCs and promoting mineralization [17]. Several in vitro investigations have demonstrated that MSCs of different origin and osteoprogenitor cells were able to produce mineral matrix, when seeded on 3 D scaffolds [18]. Interestingly, composite of ECM protein Collagen and HA, or TCP, have been recently used to produce scaffolds and have shown to trigger and accelerate osteogenic differentiation and support in vitro bone formation from MSCs [19, 20] .

However the efficacy of a scaffold in generating in vitro bone formation could not be accompanied by the same properties in vivo, thus, to translate the technology of engineered scaffolds into clinical applications, in vivo approaches through animal models are necessary.

Different scaffold materials for cartilage engineering have been tested, they can be natural derivatives, synthetic polymers or a mix; they can present different forms: solid in fibers, powder, mesh, sheets, semi-solid gel, hydrogel, or glue form [21].

Several types of materials have been used for scaffolds manufacture. Some may contain proteins such as collagen, fibrin or gelatin, others may be made of carbohydrates (hyaluronic acid, alginate, agarose, polyglycolic acid (PGA) or poly-lactic-acid (PLA) and chitosan).

Chondrocytes have been seeded on biological matrices such as hyaluronic acid [22, 23] or collagen membranes [24]. These latter are the most widely used and have been studied for the first time in 1984 [25]. Collagen, as recognized by enzymes, can be modeled and degraded over time [26], it can induce transplanted cells to produce more Collagen [27]. Scaffolds constituted of commercial type I/III collagen membranes have been introduced [28].

Among the commercial products currently used for cartilage regeneration, there are two poly (lactic acid) (PLA) scaffold-based systems in particular: Bioseed ®-C and TRUFIT CB™ [29].

Bioseed ®-C from BioTissue Technologies is a scaffold of PGA/PLA and polydioxanone (PDO) on which autologous chondrocytes are cultivated. This is a method similar to the Autologous Chondrocyte Implantation (ACI); more than 3000 patients have been treated with it since 2002 and it is available in Europe [30].

TRUFIT CB™ from Smith & Nephew, differently from Bioseed ®-C, is a system similar to the Osteoarticular Transfer System (OATS), that is a replacement of damaged cartilage with cartilage taken from a site which is subject to a lower weight. This scaffold is composed of a poly-(D-L-lactide-coglycolide) (PDLGA) and calcium sulfate bi-layer [31].

There is a great variety of products for cartilage engineering, many of which are in the clinical study phase. Most of these scaffolds requires the use of passaged chondrocytes, only in few cases it is possible to co-culture primary chondrocytes and less differentiated cells such as stem cells (in particular MSCs).

Primary chondrocytes can be enzymatically isolated digesting biopsies from less loaded regions of the knee. In order to obtain an adequate number of cells, chondrocytes need to be passaged one to four times, but it is important to maintain them at a low passage to avoid their dedifferentiation [32].

During chondrocytes differentiation there is a morphology change, the cells become round fibroblast-like at the same time specific cartilage matrix production is reduced. These changes are accompanied by a down regulation of chondrogenic genes and an up-regulation of fibroblast or mesenchymal genes [33, 34]. All these changes occur, generally, in the first passages. The passage number refers to the number of times in which the cells are passed in monolayers in culture and it appears to have little effect on the cells ability of new matrix synthesis. Generally it is preferable to use cells passaged less than 4 times [35].

Chondrocytes can be cultured in bi-dimensional cultures in the presence or not of serum, antibiotics and antimycotics. Advantages and disadvantages related to the use or not of these supplements are still under investigation since there are many conflicting data. Some growth factors, such as FGF-2, EGF and TGF-β1, appear to be indispensable to increase cell proliferation and enhance chondrogenic phenotype [36].

A particularly attractive strategy for improving the regenerative cartilage processes, compared to the use of differentiated cells such as chondrocytes, is represented by progenitor cells, in particular MSCs, with a chondrogenic differentiation potential [37, 38].

To obtain a correct hard tissues regeneration, MSCs have to be harvested, collected and isolated with opportune techniques. MSCs have been discovered by Friedenstein et al. who described a population of nucleated adherent cells from bone marrow that were fibroblast-like and capable to differentiate into fibroblast, osteoblast, chondrocyte and adipocyte [39].

In general, MSCs represent a small fraction of cells in the bone marrow, thus the frequency of MSCs in human bone marrow has been estimated to be in the order of 0.001–0.01% of total nucleated cells [38].

A higher percentage of MSCs can be isolated from dental pulp, the tissue harvested from a single tooth and following the opportune treatment can generate a large number of colonies considering the small volume of the tissue [40].

The identity of these cells as MSCs has been confirmed by their ability to differentiate also into neural-like cells, adipocytes, odontoblasts, and myoblast cells confirming their multipotency [41].

Such important features have led to define these cells as Dental Pulp Stem Cells (DPSCs).

These cells are located in the soft tissue within the tooth, in a niche of the pulp chamber mixed with the others cells normally present in the connective tissues and surrounded by the odontoblasts. The resident cells are fibroblasts, macrophages and granulocytes.

DPSCs can undergo to proliferation, allowing their self-renewal but also to differentiation process forming new odontoblasts and repairing the dentin damaged by bacterial action during caries [42].

DPSCs can be easily isolated from permanent and deciduous teeth; DPSCs are multipotent and have an high proliferation rate and the ability to undergo both osteogenic and chondrogenic differentiation.

Thus they have the potential to be used in the treatment of bone and cartilage injury and trauma having osteogenic and chondrogenic features and high proliferation rate [43,44,45]. MSCs, capable to differentiate into osteoblasts and chondrocytes, can be isolated not only from dental pulp but also from other dental tissues such as periodontal ligament, apical papilla and alveolar bone; anyway the dental bud which is the precursor of the tooth, or part of it as the dental follicle, are getting high interest as MSCs source [46]. The dental bud of the wisdom tooth can be harvested in teenagers and represents a productive source of MSCs. This undifferentiated organ contains an high number of stem cells, most of them expressing mesenchymal makers and, opportunely treated, can express excellent osteogenic features, representing an excellent model for bone regeneration (Fig. 5.1) [47]. Cartilage regeneration with MSCs is still a step behind bone regeneration. It is known that MSCs biologic processes are controlled by WNT/Beta-catenin pathway, which promotes osteoblastic differentiation during MSCs growth in culture. Therefore in normal conditions MSCs decrease the chondrogenic potential. Thus the inhibition of WNT signaling together with FGF2 administration during differentiation can promote chondrogenesis [48]. Also gene therapy showed to be effective in increasing chondrocyte differentiation from MSCs [49]. Chondrocyte obtained from MSCs differentiated on scaffolds of resorbable composite of natural hyaluronan matrix and synthetic polyglycolic acid showed to be more successful in repairing microfractures and large chondral defects in humans [50].

Fig. 5.1
figure 1

(A) DBSCs isolated from dental bud and cultured in vitro. The cells were able to adhere to the plastic and after 48–72 h acquired the typical colony-forming unit appearance. (B) DBSCs cultivated in presence of mineralizing medium for 21 days and stained for Alizarin Red. Dense nodules stained in red are visible (40× magnification)

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5.1 In vivo Models of Bone Tissue Repair and Regeneration

5.1.1 Ectopic Bone Formation

Ectopic bone formation represents the ossification of tissues in not usual places. This event is pathologic and has a clinical relevance, but has been used as a model to test in vivo experimental bone formation starting from cells and scaffolds. Subcutaneous implantation is the most used because surgically easier and is applied especially in rodents for three valid reasons: they are less expensive, have soft skin and can be readily immunocompromised for xenograft-based experiments. Basically, skin incisions are made on the dorsum of the animals under aseptic conditions and subcutaneous pockets are created where implants are placed. The most common implanted cell types are MSCs from bone marrow (BMMSCs), but experimental models have been also developed with DPSCs, Periodontal Ligament Stem Cells (PDLSCs), cord blood MSCs and osteoprogenitor cell lines [51]. Very few studies reported ectopic bone formation from direct transplantation of free-scaffold cells [52], few more revealed in some cases the ability of free-cell scaffolds to induce bone formation, but mostly in presence of BMP-2-loaded composite materials [53, 54]. Whereas the majority of the studies showed the best results when the cells were delivered with the help of different types of supports, in most cases the cells, after culture expansion, were seeded on biomaterials and predifferentiated in vitro with osteogenic factors before implantation [51, 55]. The use of immunodeficient animals, especially rats and mice, allow the implantation of allogenic or xenogenic cells with no adverse effects; the use of human cells, in particular, may have more important clinical implications. The subcutaneous experimental model is accomplished with small size rodents; the advantage of using small size animals consists in an easier accessibility for the scientists, allowing evaluation of bone regeneration and repair in relatively short time and with a high number of samples (implants) coming from the same animal. Furthermore in this model is examined the bone formation deriving from cells not naturally present in the subcutaneous, thus when xenogenic stem cells are implanted, it is confident enough to establish that the new formed tissue is of exogenous origin and not from native derivation. This latter aspect is especially important when implants with human stem cells are tested and has important clinical relevancies; however, it should be noted that this model does not take into account the influence of bone microenvironment and mechanical loading, thus restricting its availment to a first phase of in vivo evaluation of a tissue regeneration system. Indeed in order to translate the acquired experimental knowledge into real clinical cases it is necessary to develop more specific bone-defect models.

5.1.2 Craniofacial Models

5.1.2.1 Mandibolar Bone Defect Model

The choice of suitable animal model to test depends on which type of bone defects are object of the clinical demand and need to be simulated in animals. The craniofacial bones are often subjected to surgery due to tumors, traumas or congenital malformations. For example, parts of mandible can be removed for oncologic causes, thus the reconstruction of this bone represents a big challenge. To this purpose mandible defect models have been generated in rat, dog, goat and monkey to study bone regeneration from stem cells with and without the presence of scaffolds. These models include different size of defects ranging from 1 mm in mice, to 35 mm in sheep and are defined critical size defects (CSD), which is a defect size that will not undergo normal repair during the lifetime of the animal [56,57,58]. To repair such critical size defects, tridimensional scaffolds are necessary in order to recreate the structure and functionality of the lost bone tissue, furthermore the integration of the new bone with the resident tissue is needed, together with the appropriate degradation of the scaffold at the end of the healing process. The most common scaffold materials used in mandibular defect models that meet these characteristics are Bioceramics with incorporated (HA) (TCP). BMSCs seeded on these type of scaffolds have shown to have good bone repair potentials in jaw defects [57, 59,60,61] followed by dental tissues derived MSCs [55, 62]. Interestingly, also injected ASCs (Adipose-derived stem cells) ameliorated the healing time and promoted bone formation [63, 64].

5.1.2.2 Alveolar Bone Defect Models

Periodontitis is a disease of the tooth-supporting (periodontal) tissues characterized by inflammation and bone loss [47] impacting on health and life quality. Focusing on repair of alveolar bone defects caused by periodontal destruction would be a significant clinical goal. Periodontal defects have been surgically generated in animals to set in vivo tissue engineering of periodontium. The majority of the studies were conducted in large animals as dogs and minipigs using autologous cells for the regeneration, while nude rodents were employed for xenogenic cell implants. The data present in the literature indicate that autologous and allogenic MSCs of dental origin, mostly dental pulp and periodontal ligament, as well as bone marrow-derived have the ability to differentiate and regenerate the periodontium tissues, including bone and cementum, with well-oriented ligament fibers [56, 65].

5.1.2.3 Calvarial Bone Defects Models

The calvarial model is particularly suitable for evaluating the regeneration ability of high complex implant or new composite materials, because of poor vascularization of the bones and low presence of bone marrow [66]. This model has been most commonly developed in rodents as rats and rabbits, but also in mice, pigs, sheep and goats. It is widely utilized because the bones shape allows to induce consistent and reproducible bone defects, easy to surgery, does not require fixation during the healing, and is easy to analyze with histological and radiological techniques after the healing period. Cranial defects generated in nude mice, were used to evaluate bone regeneration capacity of scaffolds made of TCP and granular deproteinized bovine bone, alone or in association with hDPSCs, indicating that addition of cells to scaffolds ameliorated the bone regeneration process [67]. Similar results were obtained in a rat model carried out with hDPSCs combined with a HA/TCP scaffold [68]. A recent study has used a calvarial defect model in rabbit to test a composite ceramic of TCP and HA in combination with rhBMP-2 and autologous MSCs, demonstrating that a certain composition of TCP and HA in synergy with rhBMP-2 and MSCs enhanced new bone formation as well as the resorption rate of the scaffold [69]. The limit of this experimental model is the absence of loading weight sites, thus in some applications others bones as mandibles, or long bones femur or tibia, may be preferred.

5.2 In vivo Models of Cartilage Repair and Regeneration

MSCs cells exhibiting trophic and immunomodulatory activities [70], could positively influence fate and activity of the unaffected cells surrounding the cartilage at the site where the damage is.

Notwithstanding the great interest in referring to MSCs, little data are available on studies with large-animal models.

As previously said for biomaterials used with chondrocytes in cartilage regeneration, MSCs can be delivered in cartilage defects through hyaluronic acid/hyaluronan, which is a glycosaminoglycan particularly abundant in the cartilage ECM, or collagen-based biomaterials [71, 72].

Kasemkijwattana C et al. have tried autologous implantation of BMMSCs, after their expansion on Collagen scaffolds, showing the advantages of this procedure compared to the convectional ACI [73].

However a problem to be solved is represented by the integration difficulty of different biomaterials with the neighboring cartilage to reach a continuity between the neo-synthesized tissue and the native one and long-term healing effects [74]. In this respect, promising results have been obtained recently in a rabbit model, using MSCs sheet incorporated in a bi-layer of Poly-lactic-glycolic acid (PLGA)/MCSs [75].

MSCs can be used also for transplantation in the damaged site after their chondrogenic differentiation in vitro. The optimal protocol for the differentiation of MSCs into chondrocytes is still under investigation since the use of the protocols available today has allowed to obtain hypertrophic cells that, when transplanted in Severe Combined ImmunoDeficiency (SCID) mice, led to the formation of an unstable cartilage [76].

An alternative is the use of chondrocytes and MSCs co-cultures. Studies carried out in this direction have led to phenotypically stable tissue constructs containing a high amount of proteoglycans and Collagen Type II. This could in the future allow to reduce the use of chondrocytes for in vivo implantation [77].

Although the methods based on scaffolds are nowadays widespread, further studies are needed to find the ideal matrix material. Even if cartilage engineering represents a promising solution, an adequate approach for the long-term regeneration of cartilage lesions has not yet been identified.

5.3 Clinical Studies

For both bone and cartilage tissue-engineering approach, to integrate opportunely differentiated MSCs with the correct scaffold would represent a promising strategy for hard tissues regeneration, generating new, cell-driven, functional tissues, rather than cell free allogenic or heterologous tissue grafts.

Since many years, clinic applied research has demonstrated a successful therapy in patients with bone defects that have received grafts with autologous and opportunely in vitro differentiated MSCs integrated with hydroxyapatite scaffolds. Hard tissue engineering exploiting MSCs differentiated in osteoblasts or chondrocytes and seeded on biocompatible three-dimensional scaffolds, allows tissue-like structures formation with vascular ingrowth.

At present different kinds of scaffolds are already used to regenerate degenerative or traumatic bone defects: both synthetic bone mineral matrix or bio-absorbable ones can be enriched with growth factors (BMPs) or platelet enriched plasma for more effective results [78]. The integration of differentiated MSCs with the mentioned scaffold could make regenerative therapies also more effective compared to autologous bone therapies. These treatments would have the great advantage to avoid autologous bone graft preserving the skeleton and the harvesting site surgical consequences [79]. Yet, there are some issues that have to be further investigated: MSCs from bone marrow are not easy to be isolated and expanded; other sources, as dental tissues and peripheral blood, appeared to be more convenient. Moreover pre-clinical studies on mice showed that MSCs used for bone regeneration could lead to ectopic bone formation [80].

Cartilage restoration instead requires a different approach and can be used in patients with small cartilage lesions. It can be accomplished using a variety of methods as tissue grafts (autografts or allografts) or techniques adequate to stimulate the natural repair process [2].

In order to eliminate the necessity of a donor site and the concerns associated with allogenic and autogenic implants, many attempts have been made to heal or regenerate the existing cartilage, rather than to replace it. The techniques suggested are focused on improving the intrinsic tissue regenerative properties or on chondrocytes transplantation with the objective of generating more tissue. Unfortunately, no one of these techniques has led to a complete success, especially in older patients.

The most common treatment of cartilage regeneration consists in penetrating the subchondral bone providing a scaffold for MSCs migration and their eventual differentiation into chondrocytes and osteocytes [81]. This method leads to a large variability in the results.

Less invasive techniques are considered laser or electrical stimulation, or pharmacological agents used to stimulate chondrocytes activity [2].

Cells transplantation, using chondrocytes or undifferentiated cells, can be used to restore the mass of cartilage tissue lost. It is necessary a small tissue biopsy, the number of cells obtained is expanded in culture and then the resulting cells are placed where the defect is present. Chondrocytes transplantation is object of studies since 1968 [82, 83].

Most promising results were obtained by using a surgical method that utilizes a periosteal flap sutured on the defect as a barrier for cultivating injected chondrocytes [84]. This procedure is called ACI and results in a filling of the defect with hyaline cartilage or mixed-type neocartilage which is integrated with the host tissue [85]. After ACI first application by Brittberg in 1987, the method has evolved [86, 87], since an issue associated with the use of a periosteal membrane was often represented by hypertrophy [88].

In order to contain differentiated cells after transplantation in a defined area and let them to distribute uniformly, it is possible to use scaffolds made of porous materials instead of tissue flaps. This technique is less invasive, reduces the morbidity related to the use of a donor site and provides an anchoring substrate which is fundamental for cell adhesion processes [89].

The researchers have tried to reconstruct cartilage tissues in vitro through tissue engineering, a technique that combines the use of cells and biomaterials providing scaffolds on which the cells can grow in three dimensions and under physiological conditions [90].

Chondrocytes cultivated on two-dimensional cultures differentiate, tend to assume a more flattened appearance and produce Collagen I in place of Collagen II. If cultured on three-dimensional systems, the cells maintain their phenotype and their functionality [91]. The 3D culture produces microenvironments very similar to those of the native cartilage, favoring the formation of cell-cell and cell-matrix interactions, this is an advantage which distinguishes the 3D from the 2D culture. An ideal scaffold should act as a support for the cells during new cartilage formation, then replaced by the neo-synthesized matrix and, above all, should be biocompatible.

Scaffolds can be also used with no cells to promote cell migration to the purpose of regeneration enhancement.