**8. Biotechnology**

affected) the healing is not obtained, even with a correct treatment and above all make possible

In the new NUSS classification of 2008 are considered all the variables and all risk factors, giving to anyone a score based on clinical experience and scientific evidences and defining so

The final score, obtained by the sum of the individual score, allows to compare different patients with different non-union, making them objectively comparable according to a

Atrophic forms of non-union can have better prognosis and greater chance of healing than oligotrophic reactive forms, in patients affected by impaired general health condition, as in

**•** The bone (quality, kind of fracture, number of previous surgical procedures, and their invasiveness, non-union classification according to Weber–Cech, adequate first surgical

**•** Soft tissues (tissues conditions, vascurarization and possible surgical procedures on soft

**•** The patient (ASA score—American Society of Anesthesiologists—diabetes, laboratorys

First group, score from 0 to 25, made mainly as mechanical problem, the treatment indicated

Second group, score from 26 to 50, made the problem as both mechanical and biologic, the treatment needs correction of the synthesis and biologic stimulation of the fracture site, obtained with the help of physical means (magnetic electro-pulsated fields, extracorporeal

Third group with score from 51 to 75. Is a complex problem characterized by high gravity of both biological and mechanical conditions? It is almost always required the resection of the non-union site, and then, is present a bone loss that have to be restored. Next to bone transport techniques with external fixator and tibiotarsal joint arthrodesis at the docking point, there is indication to autologous bone transplant and biotechnologies (cells, scaffolds, and growth factors) applied in polytherapy according to the principles of the "Biological Chamber" [23–

the drafting of a therapeutic choice algorithm [18, 19].

a treatment guideline depending from the final score [17].

**7. The variables considered are as follows:**

exams, infective condition, drugs, and smoke).

procedure in order to mechanical stability, bone gap, alignment);

is the fracture stabilization, optimizing or changing the synthesis system.

shock wave) or with the application of biotechnologies in monotherapy [20–22].

**6. The NUSS**

112 Advanced Techniques in Bone Regeneration

principle of complexity.

example a non-compensated diabetes.

tissues and skin coverage);

25] [case 1].

Biotechnology at our disposition are synthetic growth factors (GFs) as human bone morpho‐ genetic recombinant proteins (rh-BMPs), autologous growth factors (AGFs) contained in platelet-enriched plasma (PRP), mesenchymal stem cells (MSCs), and scaffolds or bone substitutes.

**•** Growth factors (GFs)

Since the second half of 1990s, it has been demonstrated that some growth factors act as powerful stimulators of the in vitro osteoblastic proliferation and of the in vivo bone healing, such as to turn out really useful in aiding the healing process if correctly applied at the site of the lesion [26]. Thanks to the evolution of the tissue engineering, it is been possible to produce the single growth factors with the recombinant-DNA technique, particularly the rh-BMPs. Although they have been identified at least 40 different rh-BMPs, a clear clinical demonstration of the osteoinductive potential is available only for the rh-BMP-7, also known as osteogenic protein-1 (OP-1), and for the rh-BMP-2 [27], belonging to the transforming growth factors family (TGF-β), whose receptors are expressed on chondrocytes and osteoblasts [28]. The osteoinduction phenomenon is characterized by the transformation of the perivascular mesenchymal cells in bone progenitor cells that can regenerate bone tissue. The recombinant human osteogenic protein-1 (rh-OP-1), also known as rh-BMP-7 (epto‐ dermina-α), conveyed by type-I collagen, has been the first to be approved in the world to treat non-union of long bones and in the USA as "humanitarian device exemption" (HDE) in the treatment of spinal non-union. It allows, also, the regeneration from vascularized bone and of healthy bone surrounding toward the inside deficient area. Thanks to several preclinical and clinical studies, the efficacy of the use of rh-BMP-7 has been demonstrated reporting in some studies success percentage between 85 and 89%; at the same time, it has been found a real decrease of complications linked to the use of autologous bone, considered even at this time the "gold standard" [29–38].

**•** Autologous growth factors (AGFs) and platelet-rich plasma (PRP)

The PRP is the most advanced product of the "blood management." It is a biologically active concentrate of mediators extracted from patient's plasma and is a source of non-specific autologuos growth factors [platelet-derived growth factor (PDGF), TGF-β1-β2, insulin-like growth factor type 1-2 (IGF1-2) and vascular endothelial growth factor (VEGF)] able to stimulate bone, cartilage, and soft tissues healing processes on the site of use. It is charac‐ terized by an elevate concentration of trombocytes able to degranulate releasing several growth factors and cytokines that can induce osteogenesis and angiogenesis with a chemo‐ tactic and mitogenic mechanism [39]. It can be obtained from autologous or heterologous blood. Depending from the procedure used to treat the withdrawal can be obtained final platelet concentration from 4 to 8 times higher from the initial situation. In a randomized study of 2007 on 60 long-bones non-union has been demonstrated a minor healing capabil‐ ities by the PRP (63.8%) both in comparison with BMP-7 than to the autograft [30].

The AGFs contained in the PRP, as clarified by preclinical and clinical data, are promoters of the cellular division (mitogenesis) nonspecific for the bone cells, unable to promote the differentiation of the mesenchymal cells and to induce the formation of new bone tissue. They seem to be not useful when used alone or in association with scaffold in treatment of tibial pilon non-union.

**•** Mesenchymal stromal cells (MSCs)

Studies based on cellular therapies are concentrated on a rare non-hematopoietic cells population, the MSCs, which are present in patient's bone marrow and can be increased in colture in an undifferentiated state [40, 41]. In addition to their pluripotent properties, the MSCs are considered osteogenic progenitor cells with demonstrated ability to repair bone defects [42]. Their concentration at bone marrow level, however, can result not ever elevated [43, 44]. The influence of this factor seems to be fundamental to the aim to obtain the healing, and there are clinical evidences that a better prognosis is obtained with a progenitor cells concentration >1500/cm<sup>3</sup> . Recently, new techniques have become available to obviate to this problem, between these patient's bone marrow aspirate permit the mesenchymal stem cells concentration directly in the operatory room. Those new methods have demonstrated two big advantages: a reduction in costs respect to the in vitro expansion of the MSCs and a drastic decrease of the donor site morbidity compared to the traditional collection in open surgery of the iliac crest [44, 45]. The clinical use of the MSCs, especially if associated with the BMPs, it has proven effective determining the non-union healing [46].

**•** Scaffold

The osteoconduction mediated by the scaffold is determined by the chemical–physical characteristics of the substratum act to favor the adhesion and the growth of the cells on the surface. The mechanical characteristics of the bone graft, and their resistance to the com‐ pression and torsion, are influenced from their shape (massive, cortical splint, spongious block, morcellized), from the withdrawal modality, processing, conservation, and from the kind of synthesis meaning used.

The synthesis substitutes used are mineral structures similar to human bone kind. They have only osteoconductive power. Between synthesis substitutes you can find calcium phosphate as hydroxyapatite, coralline hydroxyapatite (absorbable), tricalcium phosphate (TCP, absorbable), and biphasic calcium phosphate (BCP = HA + TCP). For small defects, the hydroxyapatite is good filler ad favorite, thanks to their osteoconductive properties, the progressive revascularization and reossification of the treated area. All materials available have some limit. Ceramic, particularly, presents three important disadvantages: the difficulty to remain in place, the long time needed to absorption, and the complete substi‐ tution with neoformed bone and the impossibility to fill important bone gap.

The allogenic transplant from bone bank and heterologous animal origin (porcine, bovine, or equine) have demonstrate osteoconductive power but not osteoinductive. They need, then, to be revascularization and repopulated from the outside, needing a surrounding enabling environment. Con be used as filler (morcellized/granules) or as mechanical support (wedges, blocks, splints) [47, 48].
