The repair of bone defects remains challenging in the treatment of conditions such as osteomyelitis, osteonecrosis, bone cysts, bone tumors, or implant failure caused by osteolysis or nonunion . Generally, bone has a very good self-healing capacity and acute fractures typically heal without additional interventions . However, for defects larger than a critical size spontaneous healing is not observed. The gold standard for the treatment of bone defects is autologous bone grafting but the amount of tissue that can be obtained from the iliac crest and other sources is naturally limited and donor site morbidity remains an important issue . Therefore, research has focused on the development of bone scaffold materials that can be implanted into a defect of a given size and would stimulate the formation of new bone tissue in the defect along with the gradual resorption of the scaffold.
Among the promising scaffolding materials, partially synthetic bone substitute materials have been developed, such as hydroxyapatite , glass-reinforced hydroxyapatite , brushite , tricalcium phosphate , and mixtures of these materials (composites) . Hydroxyapatite ceramics and cements represent the most important clinical scaffolds. The structure of such materials is characterized by a system of interconnecting pores that induces osseous integration . In addition, bioceramics can be seeded with mesenchymal stem cells for regenerative therapies to utilize their proliferation capacities and abilities to build new tissue in a defect .
Biodegradable polymers represent a promising class of materials designed to match the mechanical properties of hard tissues. The advantage of polymer structures is that they can be chemically tailored to be resorbable in a time frame appropriate for defect healing and remodelling . The most frequently applied biodegradable biopolymers are poly(D,L-lactide-co-glycolide) (PLGA) scaffolds. These materials are approved by the U. S. Food and Drug Administration for medical use both experimentally and clinically . PLGA polymers degrade in the presence of water to lactic and glycolic acid, which both represent natural by-products in several metabolic pathways. Although these acidic degradation products may not be ideal as they can cause local inflammation, these materials have been successfully implanted in bone defects numerous times and their compatibility and biosafety have been proven [11, 13, 14]. Typical fabrication techniques for PLGA scaffolds further allows structural modifications, for instance to adjust the surface of the grafts for optimal cell-biomaterial interactions or to blend the materials with mineral particles to adjust mechanical properties and bioactivity [15, 16]. Porosity and pore size of the material can be controlled as they play a crucial role in bone formation . The pore size of the materials can be adjusted to provide optimal conditions for cells to colonize the scaffolds and produce the extracellular matrix (ECM) for optimal osseous integration. Therefore, we studied the influence of the pore size of the PLGA scaffolds on healing of a tibia defect in rats.
In addition to standard histological examination, we applied a comprehensive set of MR techniques and mass spectrometry (MS) for the analysis of the de novo bone formation in the implanted scaffolds. NMR spectroscopy is capable of quantitatively monitoring the development of the major components of the organic (∼20 wt % of bone) and inorganic bone matrix (∼70 wt %), in particular collagen and hydroxyapatite [18, 19]. In addition, NMR spectroscopy allows for comparison of the molecular structure and dynamics of the de novo synthesized molecules in the implants with those in the natural tissue. Thus, a quantitative and atomistic picture of the essential molecular properties of the bone tissue in the implants can be provided . In particular, the molecular dynamics of collagen is a sensitive marker for the degree of mineralization and the state of the development of the bone tissue . While previous NMR studies could only be carried out in isotopically enriched tissues , modern techniques now allow for the detection of the collagen moiety in natural tissues already at natural abundance of useful NMR isotopes (particularly 13C) [22, 23] enabling a quality assessment of the de novo formed bone even from a biopsy without any further sample treatment. Although various MR parameters had been shown to be dependent to the cartilage glycosaminoglycan (GAG) content [24, 25], MALDI-TOF (matrix-assisted laser desorption and ionization time-of-flight) MS is particularly sensitive to the GAG of bone, which can be studied quantitatively [26, 27].
The aim of this study was to analyze the properties of the de novo formed bone and ECM in PGLA scaffolds of different pore sizes at 2 and 4 weeks after implantation into the rat tibia using histology, MRI, quantitative NMR spectroscopy, and MALDI-TOF MS.
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PLGA scaffolds are widely applied in regenerative medicine [11, 40-42]. However, although the pore size of the material is of crucial importance for cell migration , studies that systematically investigate the pore size-dependence of polymer-based implants for bone regeneration are scarce [44, 45]. To the best of our knowledge, only a single study exists using similar macroporous PLGA scaffolds , but a completely different model (mesenterial tissue) has been used and the middle pore size has not been studied at all.
We applied a comprehensive analytics including histology, MRI, MALDI-TOF MS as well as 13C and 31P MAS NMR spectroscopy. Although NMR is a versatile and quantitative tool, it does not represent a standard technique for the application of the regenerated ECM in bone in spite of the obvious advantages of the method [22, 34-36, 47, 48]. Apart from the analytical and structural power of NMR spectroscopy with regard to the qualitative and quantitative assessment of de novo generated tissue, the NMR techniques also allow the study of the dynamics of the biopolymers. NMR studies have highlighted the impact of the molecular mobility on the macroscopic elastic properties for polymer-based materials . In agreement with this aspect, the molecular dynamics of the ECM are also related to the elastic properties of bone. Thus, the application of NMR techniques for the characterization of de novo generated tissues in implants as well as the function of the related tissues is promising [39, 50].
This study investigated the de novo bone formation in PLGA implants of varying pore size using an established rat tibia model. Taken together, the results obtained indicated that the PLGA scaffolds are osteoconductive and induce bone formation and biomineralization in these untreated scaffolds. Histological evaluations of the samples 4 weeks after implantation revealed that the implanted PGLA scaffolds were homogeneously and completely invaded by newly formed lamellar bone, while the control defects were filled only partially with less mature woven bone with strands of fibrous tissue and patches of fibrocartilage characteristic for the earlier stages of enchondral ossification (Fig. 1). The good integration of the implants was confirmed by MRI (Fig. 2), although the microscopic bone structure in the implants was isotropic and similar to the cancellous bone in the host metaphyseal bone. Some MR images, particularly in the untreated defect, revealed a morphological structure at the perimeter of the defect that is reminiscent to the epiphyseal growth plate in the host bone. The resemblance of the de novo formed bone to cartilaginous tissue could also be confirmed by quantitative measurement of GAGs in the implants by MS, since bone formation can be described by a time-dependent dispersion of the different tissues . Fibrous tissue is the most abundant initial component but its contribution decreases during the healing process. Furthermore, in the intermediate phase, a high amount of cartilage is present  providing an order of magnitude higher GAG content in comparison to bone . Consequently, in the initial state, higher GAG content as in native bone is observed, while the amount of GAG decreases during a normal healing process as confirmed by MS (Fig. 7).
The formation of organic and inorganic ECM increased with time. On an atomistic level, NMR could confirm that both collagen and hydroxyapatite were generated in the scaffolds. The structure, molecular concentration, and dynamics of these de novo synthesized molecules were found to be similar to that of the native bone. Collagen type I represents the largest fraction of organic molecules in bone and its ECM . Using 13C MAS NMR, collagen could be unequivocally detected in the implants. As the isotropic chemical shift of a carbon peak is indicative of its secondary structure  and the chemical shift of the de novo formed collagen in the implants agree with those of the bone collagen, we conclude that the characteristic triple helix structure of collagen had been formed in the PLGA implants. Interestingly, the majority of 75–95% of the collagen has already been synthesized within the first 2 weeks, while collagen synthesis was apparently downregulated between the 2nd and 4th week. Unfortunately, our quantification procedure did not allow an absolute measure of the collagen content. However, the reduced synthesis in the second half of the experiment suggests that the collagen content already reached its maximum after 2 weeks.
In contrast to collagen, the majority of the bioapatite was synthesized after 2 weeks. Biomineralization takes place after collagen is synthesized and the hydration water of collagen is then replaced by the bioapatite crystals . It has been reported that the interaction of collagen with the mineral may restrict the motions in collagen . This effect has also been observed here as the motional amplitudes of the collagen motions are typically higher after 2 weeks of implantation, which indicates that the collagen segments can undergo larger amplitude motions. Higher order parameters (i.e., smaller amplitude motions) were observed in the de novo formed collagen after an implantation time of 4 weeks, which are comparable to those of native bone. However, it should be noted that the collagen motions in the implant are much more restricted than in fully hydrated cartilage  suggesting that the newly synthesized and less mineralized collagen also experiences a significant motional restriction.
From the analytical results of our study, we conclude that the de novo formation of bone in the tibia model is already nearly complete after 4 weeks. Bone formation was most pronounced for the middle pore size of 300–500 μm. This does not appear to be a trivial effect of the geometrical parameters of the scaffolds as densities and hence porosities of the scaffolds were rather homogeneous [85.7%, 87.3%, and 87.3 % porosity for the pore sizes of 100–300, 300–500, and 500–710 μm, respectively ].
It is easily comprehensible why the largest pore size did not provide an optimal performance. Here, the pores are too large to provide optimal adhesion properties for the cells to migrate into the defect and fill up the empty space. The situation is then almost comparable to a free 2D cell culture that is known not to grow into the third dimension. In addition, such scaffolds would be most compromised with regard to stability. The smallest pore size also showed inferior performance. Although these pores exceeded the smallest recommended pore size for ceramic materials of 100 μm , literature data have also shown better osteogenesis for hydroxyapatite and titanium alloy implants with pores > 300 μm [58, 59]. While pores of 100–300 μm in PLGA materials are still much larger than the cell dimension, it is conceivable that the wetting properties of this material are compromised. Although we wetted the scaffolds via an ethanol phase to overcome the hindered entry of water into the air-filled pores, the procedure might not yield perfect results.
In conclusion, our study demonstrated the use of MR techniques and MS for the assessment of de novo bone formation in polymer-based scaffolds in an animal model. It is quite remarkable that the de novo formed bone ECM in PLGA implants quantitatively and qualitatively closely resembles that of healthy native bone according to the parameters provided by these methods. A pore size of 300 to 500 μm provides the best results regarding the formation of bone and ECM and, therefore, represents the most effective implant. Furthermore, the PLGA implants proved to be osteoconductive as shown by the generation of bone-like ECM within the PLGA treated defect as oppose to the untreated defect. It is well known that high porosity and large pores facilitate bone ingrowth and osseointegration into a scaffold after implantation . However, if the pore size exceeds a critical value, mechanical stability of the scaffold is diminished. Furthermore, cell migration in scaffolds with large pores may also be limited. Consequently, PLGA implants with 300 to 500 μm pore size appear to be attractive matrices for further studies that will focus on biofunctionalization of those scaffolds, e.g. with ECM components and growth factors.