PRP-Augmented Scaffolds for Cartilage Regeneration: A Systematic Review

PRP-Augmented Scaffolds for Cartilage Regeneration: A Systematic Review Elizaveta Kon, MD,* Giuseppe Filardo, MD,* Berardo Di Matteo, MD,† Francesco Perdisa, MD,† and Maurilio Marcacci, MD† Modern regenerative procedures for articular cartilage defects have proved to provide good replacement of damaged cartilage, although, at present, the properties of a native healthy cartilage are still not achievable by any substitute. Several scaffolds have been tested and clinically used over the years to help the restoration of articular surface, some of them producing a hyaline-like reparative tissue. Concurrently, biological strategies are used more extensively, alone or in combination with scaffolds, to enhance the clinical outcome in patients with chondral disease. Among these innovative methods, one of the widest used is plateletrich plasma, with the rationale of taking advantage of the huge amount of GFs contained in platelets to promote cartilage regeneration. The aim of the present manuscript is to review systematically the current evidence in preclinical and clinical practice concerning platelet-rich plasma–augmented scaffolds to treat cartilage disorders. Oper Tech Sports Med 21:108-115 C 2013 Elsevier Inc. All rights reserved. KEYWORDS platelet-rich plasma, cartilage, regenerative medicine, scaffolds

T

Scaffolds: Definition and Application

*Nano-Biotechnology Laboratory, Rizzoli Orthopaedic Institute, Bologna, Italy. †Biomechanics Laboratory, Rizzoli Orthopaedic Institute, Bologna, Italy. Address reprint requests to Elizaveta Kon, MD, Nano-Biotechnology Laboratory, Rizzoli Orthopaedic Institute, Via di Barbiano n. 1/10, 40136, Bologna, Italy. E-mail: [email protected], [email protected]

Articular cartilage is a unique tissue with poor regenerative potential. Although over the years many different approaches have been clinically used to treat cartilage disease, the properties of native hyaline cartilage are still irreplaceable by any repair tissue, thus its surgical management is still controversial. Among tissue-engineering methods, several biomaterials in different physical forms (fibers, meshes, and gels) have been considered for cartilage regeneration.4 Whereas solid scaffolds provide a 3-dimensional (3-D) substrate that cells can adhere to, gel scaffolds physically entrap the cells themselves. Materials range from molecules found in the cartilage matrix to synthetic substances, such as polylactides (polylactic and polyglycolic acids). Natural materials include hyaluronic acid (HA), collagen derivatives, agarose, alginate, fibrin glue, and chitosan. They are biocompatible and enhance cell proliferation. Synthetic biomaterials are easier to handle regarding their mechanical properties and degradation; therefore, it is easier to fit the scaffold to desired properties. The effect of the degradation products on the native tissue and implanted cells is still under investigation, but the biocharacteristics and biocompatibility are quickly improving.4,5 Tissue-engineered constructs enable cells to be loaded onto 3-D scaffolds that can support adhesion, proliferation, and

he approach to cartilage defects has been totally revolutionized over the past 15 years from the classic idea of just repairing damaged tissues to the innovative concept of tissue regeneration.1-3 To achieve this ambitious target, new techniques have been developed over time: the implementation of tissue engineering into cartilage-regeneration procedures has allowed bioengineered devices to be produced that can guide the regeneration of damaged tissues. Moreover, the possibility of an additional biological augmentation to improve and accelerate the healing process is under extensive investigation at both preclinical and clinical levels. The addition of growth factors (GFs) that are able to guide and promote cell growth and differentiation is gaining increasing interest, and many orthopaedic surgeons are adopting biological and bioengineered treatments. The aim of this systematic review is to analyze the real contribution of platelet-rich plasma (PRP), an easy and cost effective way to obtain many GFs in physiological proportion, as an augmentation for scaffold-based treatments of cartilage lesions.

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PRP-augmented scaffolds for cartilage regeneration matrix deposition of a hyaline-like tissue,6 and the good results of matrix-assisted autologous chondrocyte transplantation have been largely documented in the literature.7,8 Because matrix-assisted autologous chondrocyte transplantation has not been approved by the Food and Drug Administration for clinical application in the United States, alternative methods that avoid manipulation of cells and their regulatory obstacles have been developed. Thus, it has been suggested that the role of scaffolds is not only to enhance tissue regeneration by delivering cells and GFs into the lesion site but also to support and promote endogenous cell differentiation as cell-free scaffolds. In fact, scaffolds may be able to promote articular cartilage regeneration by themselves by uptaking the self-regenerative potential of the patient.9,10 More recently, new polyphasic scaffolds have been developed11,12 to treat the entire osteochondral unit and not only the cartilage layer with preliminary good results, even without the use of complex and expensive cell-based procedures. The new trend toward cell-free approaches explains the marked interest in the potential of platelet concentrates to increase tissue regeneration provided by scaffolds with a simple, 1-step, and less expensive procedure.

PRP in Orthopaedics: Biological Rationale, Definition, and Sources of Variability Blood derivatives, in particular PRP, as a biological stimulus to enhance cell proliferation by supplying endogenous GFs have been applied to several different tissues such as cartilage, tendons, and muscle.13 The biological rationale for using PRP in orthopaedics is that several GFs and other molecules contained in platelets promote healing and regeneration of the tissues and are also involved in articular homeostasis, including cartilage growth and preservation. Platelet GFs include platelet-derived GF (PDGF), transforming GF (TGF-b), platelet-derived epidermal GF, vascular endothelial GF, insulin-like GF-1, fibroblastic GF, epidermal GF, and others.13-15 Alpha granules also contain other molecules such as cytokines, chemokines, and other proteins, which are involved in chemotaxis, cell proliferation and differentiation, and inflammatory response.13 Dense granules contain adenosine diphosphate, adenosine triphosphate, Ca2þ, histamine, serotonin, and dopamine, which are also involved in modulating homeostasis and regeneration of the tissues.16 Lysosomal granules store acid hydrolases, cathepsin D and E, elastases, and lisozyme,17 and other molecules are also involved in tissue regeneration, although the mechanism of action has not yet been elucidated. Owing to the complexity of the composition of platelet concentrates, the definition of PRP itself is still unclear, mainly because of the high variability that makes comparison among blood-derived products very difficult: the use of different PRP formulations has been reported and wide interproduct variability can hinder comparisons and precise study.18

109 For example, even if a univocal definition of PRP has been proposed as ‘‘a blood derivate characterized by a higher concentration of platelets’’ (at least 200% more than peripheral blood platelet count),19,20 the literature reports a wide variability in the concentration range, up to 8 times the basal level.19 This can be considered a significant variable when comparing different PRP preparation methods, as different platelet counts may result in different amounts of GFs in the treatment site. Studies reporting correlations between clinical outcome and platelet count seem to support this issue,20,21 although the topic is still debated. One more variability factor is the presence of other bioactive molecules in the plasma, but their role still has to be clarified. Moreover, many other heterogenous and controversial variables still have to be understood, such as the presence of leukocytes, the activation methods, the storage procedures, as well as administration protocols etc. Finally, the most appropriate indication is still to be defined: several studies report the intra-articular treatment of a wide range of chondral diseases, from chondropathy to early osteoarthritis (OA) to severe OA. Findings suggest that PRP can produce different effects with relation to the patients’ characteristics.22 Thus, it appears clear that the use of PRP as an augmentation procedure for scaffold implantation might also have different results depending on the scaffold itself, as well as the characteristics of the patients and cartilage lesion treated.

Materials and Methods A systematic search was performed on the PubMed database according to the following inclusion criteria: (1) papers published in English; (2) dealing with animal and clinical application of PRP-augmented scaffolds used to treat cartilage pathology; and (3) with level of evidence from I-IV. To this purpose the following formula was used: (PRP OR platelet gel OR platelet concentrate OR platelet lysate) AND (scaffold OR implant) AND (cartilage OR chondropathy OR osteoarthritis OR chondral). All the papers found were screened to identify clinical and preclinical studies. Papers found by screening the reference lists were also considered for the literature analysis of this review (Fig. 1).

PRP-Augmented Scaffold: Preclinical Evidence PRP and biocompatible scaffolds are two relevant topics in the field of musculoskeletal regenerative medicine. The application of such biotechnologies in preclinical and clinical practices is quite recent, and as their therapeutic aim is similar, that is, promoting tissue regeneration, PRP and scaffolds soon joined forces to provide ‘PRP-augmented scaffolds,’ which are the natural consequence of several studies investigating both the properties of platelet-derived GFs and those of scaffolds. In particular, the administration of platelet-derived GFs as an augmentation for bioengineered

E. Kon et al.

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Figure 1 Research Flow-Chart for the systematic review. (Color version of figure is available online.)

chondral and osteochondral scaffolds has been tested in a number of studies (Table l), with the first experiments performed more than 10 years ago. Research has constantly moved forward in recent years, and new methods, from soaking to more complex electrospinning techniques,23 have been tested to add PRP to different kinds of scaffolds. Besides its application as an augmentation procedure, PRP itself has been manipulated to become a scaffold with the purpose of vehiculating cells and providing biological stimulation at the same time: low immunogenicity and optimal biocompatibility, together with the clotting properties of PRP, make this product an interesting carrier for tissue engineering.24 Qi et al.25 tested autologous PRP vehiculated by a collagen matrix for the treatment of patellar groove osteochondral lesions in the rabbit knee; they achieved better results both histologically and mechanically than with collagen matrix alone. A further trial by Sun et al.26 assessed the contribution of PRP added to a microporous polylactic-glycolic acid (PLGA) scaffold to treat osteochondral defects created in the patellar groove in the rabbit model. This PRP-augmented scaffold was tested against the scaffold alone and results were quite

significant: 12 weeks after the implantation, the rabbits were killed and histologic sampling revealed that in the PRP-PLGA group, the defects were completely filled with regenerated tissue similar to normal hyaline articular cartilage, well integrated with the surrounding native tissue. Even at a subchondral bone level, it was possible to observe the formation of a continuous layer of trabecular bone, which was also clearly visible at micro–computed tomography evaluation. Conversely, in the PLGA scaffold-alone group, a complete reabsorption of the implanted biomaterial was found, and the repair tissue was mainly fibrocartilage with poor extracellular matrix. Subchondral bone formation was also significantly lower than that of the PRP-PLGA scaffold group. Despite these encouraging findings, another trial showed less promising results when adding PRP to a different type of scaffold. In particular, Kon et al.27 tested a 3-layered collagenhydroxyapatite biomimetic scaffold with and without the addition of PRP. This particular scaffold was conceived with the aim of stimulating regeneration of the entire osteochondral unit, consisting of both the subchondral bone and the overlying cartilage; for this purpose, a different blend of collagen and hydroxyapatite was used in each layer. The sheep

PRP-augmented scaffolds for cartilage regeneration

111

Table 1 Animal Studies on PRP-Augmented Scaffolds Authors 25

Qi et al.

Journal and Year Cell Transplant, 2009

Animal Model Rabbit

Biomaterial Bilayer collagen matrix

Study Groups – – –

Sun et al.26

Int Orthop, 2010

Rabbit

Microporous PLGA scaffold

– – –

Kon et al.27

Lee et al.28

Marmotti et al.30

BMC Musculoskelet Disord, 2010

J Control Release, 2012

Knee Surg Sports Traumatol Arthrosc, 2012

Sheep

Rabbit

Rabbit

Collagenhydroxyapatite 3-layered scaffold

–

Gelatinpoly(ethylene glycol)-tyramine hydrogel scaffold

–

Hyaluronic acid membrane

–

– –

–

– – – – 31

Xie et al.

Biomaterials, 2012

Rabbit

PRP-derived 3-D scaffold, with meshlike microstructure

model was selected to perform the experiment, which involved creating osteochondral lesions in both femoral condyles. The aforementioned scaffold was implanted by a press-fit technique and the animals were killed 6 months after the surgical procedure. Gross specimen, histologic, immunohistochemical, and microradiographic evaluations revealed a better performance for the scaffold alone when compared with the PRP-soaked scaffold. Based on these results, the authors noted that the theoretical advantage provided by plateletderived GFs has still to be fully proven by in vivo studies. In fact, in the particular model tested, the addition of PRP was detrimental and led to a lower quality of tissue repair, and therefore the authors decided to apply the scaffold alone in clinical practice, and they achieved a good short-term clinical outcome.10,12 Each particular biomaterial might respond differently to biological augmentation, and further studies are needed to determine which factors are involved in scaffoldbased tissue regeneration and in the interaction with platelet concentrates.

– – – –

PRP þ collagen matrix Collagen matrix alone Empty defect PRP þ PLGA scaffold PLGA scaffold alone Empty defect

Scaffold þ PRP Scaffold þ cultured chondrocytes Scaffold alone

PRP þ scaffold þ cultured chondrocytes Scaffold þ chondrocytes PRP þ Scaffold þ fibrin glue þ cartilage fragments PRP þ Scaffold þ cartilage fragments PRP þ Scaffold þ fibrin glue PRP þ Scaffold Empty defect Empty defect Scaffold alone Scaffold þ BMSCs Scaffold þ ADSCs

Main Findings Better histology and mechanical results for PRP þ matrix experimental group vs collagen matrix alone. Complete filling of the defects with hyaline-like tissue, good integration, and more newly formed trabecular subchondral bone for the PRP þ PLGA scaffold group. Best gross morphologic, histological, immunohistochemical, and micro-CT quality of the regenerative tissue in the scaffold-alone group. Better results for hydrogel þ PRP þ chondrocytes: more proteoglycan synthesis, differentiation, and maturation of cocultured cells. Good overall results, best for scaffold and PRP seeded with cartilage fragments. Negative effect of fibrin glue on regeneration.

Better results both in vitro and in vivo for BMSC-seeded scaffold compared with ADSCs-seeded scaffold and scaffold-alone groups.

More recent studies further investigated the role of PRP as a biological enhancer for scaffolds. Lee et al.28 tested the effects of PRP addition to a gelatin-poly(ethylene glycol)-tyramine– based scaffold. The authors created a full-thickness defect in the rabbit femoral groove and employed 4 different scaffold prototypes to fill the aforementioned lesion: hydrogel alone, hydrogel seeded with chondrocytes, PRP-augmented hydrogel, and lastly, hydrogel þ PRP þ chondrocytes. The best results were obtained in the last group: in particular, PRP contributed to increasing proteoglycan synthesis and also played a role in stimulating differentiation and maturation of cocultured cells. Moreover, according to in vitro evaluation, PRP seemed to influence gene expression by upregulating important genes involved in the process of tissue healing, such as aggrecan, type II collagen, ChM-1, and CB.28,29 Marmotti et al.30 reported the results of an animal trial where trochlear osteochondral lesions were created in rabbit knee and treated with the implantation of a scaffold consisting of hyaluronic acid, fibrin glue, and PRP, with or without the

E. Kon et al.

12 52 No Pridie perforations þ polyglycolic-HA scaffold þ PRP Knee Clin Orthop Relat Res, 2012

Dhollander et al.33

Siclari et al.34

Knee (patella) Case series

Case series

24 5 No

No

MSCs þ PRP þ HA membrane (or collagen powder) Microfracturesþ collagenbased scaffold þ PRP Knee

J Bone Joint Surg Am, 2010 Knee Surg Sport Traumatol Arthrosc, 2011 Buda et al.37

Case series

Talus Comparative study Injury, 2010 Giannini et al.36

Giannini et al.

35

24

24

81 (25 MSCs scaffold vs 10 open ACI vs 46 arthroscopic ACI) 20 Yes (historical controls)

24 No

Scaffold made of: MSCs þ PRP þ HA membrane (or collagen powder) MSCs þ PRP þ HA membrane (or collagen powder) Talus Case series

Level of Evidence Journal and Year Authors

Table 2 Clinical Application of PRP-Augmented Scaffolds

A few studies report the application of PRP-augmented scaffolds into clinical practice (Table 2). The first surgical application of PRP was reported by Sanchez et al.32 for a cartilage avulsion in a 12-year-old football player where the fragment was reattached and the PRP was added surrounding the fragment. The patient was back to playing sport in 18 weeks at the previous level and the structural integrity of the reparative tissue was noted to be good at magnetic resonance imaging (MRI) control scans. Dhollander et al.33 reported 5 symptomatic patients with osteochondral defects of the patella treated with a collagen I-III scaffold membrane after microfractures. The interface beneath the membrane and the microfractured subchondral bone was filled with PRP. The clinical outcome at the final follow-up of 24 months was satisfactory for pain and function, in association with optimal MRI findings of the repair tissue, but at the same time, the authors could not clearly demonstrate that PRP augmentation was responsible of improving the quality of tissue repair. One more study by Siclari et al.34 described the use of a polyglycolic acid or hyaluronan scaffold augmented with PRP to treat knee chondral defects on 52 patients, evaluated 12 months after arthroscopic scaffold-augmented microfracturing: a significant improvement in the Knee Injury and Osteoarthritis Outcome Score was reported. Moreover, arthroscopic second-look biopsies showed an integrated and homogeneous, regenerative tissue with hyaline-like appearance. Giannini et al.35,36 reported the first clinical trial of bone marrow–derived mesenchymal stem cells (BMDCs) combined with PRP and either porcine collagen powder or HA membrane as a scaffold. A novel one-stage arthroscopic

Lesion Site

PRP-Augmented Scaffolds: Clinical Evidence

Clin Orthop Relat Res, 2009

Combined Treatments

Control Group

48

Patients

Followup (mo)

Outcome

addition of minced articular cartilage harvested during the same surgical procedure. Histologic scoring revealed better results for the scaffold loaded with minced cartilage, which determined the formation of a well-integrated, hyaline-like repair tissue. Another study authored by Xie and colleagues31 focused on the application of a 3-D PRP-derived scaffold with a mesh-like microstructure. The scaffold was seeded with bone marrow mesenchymal stem cells (BMSCs) or adipose-derived mesenchymal stem cells (ADSC) to test their in vitro chondrogenic potential. BMSC showed a higher proliferation rate and a higher expression of cartilage-specific genes and proteins compared with ADSC. The authors then applied these scaffolds to the rabbit model to treat osteochondral defects created in the patellar groove. Even in this case, the BMSC group gave the best results at both histologic and micro– computed tomography evaluations, with a good integration of the repair tissue within the surrounding native cartilage, a hyaline-like aspect, and a higher concentration of type II collagen. Subchondral bone regeneration was also better in the BMSC group with respect to ADSC group. Owing to the experiment design of the last 2 studies, it is not possible to assess the real contribution of PRP to tissue regeneration.

Significant increase in all the clinical scores Results comparable to arthroscopic ACI, but lower costs Both clinical and MRI good results Significant clinical improvement þ positive MRI findings Good clinical outcome þ hyalinelike cartilage at biopsies

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PRP-augmented scaffolds for cartilage regeneration procedure was used to treat osteochondral defects of the talus in 48 patients, evaluated prospectively up to 24 months after surgery; the American Orthopaedic Foot and Ankle Society score increased significantly and most of the patients were back to playing sports within 11 months. The authors found that bigger lesion size and previous surgeries were correlated with a worse clinical outcome. These findings were comparable to those obtained by autologous chondrocyte implantation (ACI) but as a single-stage procedure, thus sparing the stress of 2 operations and consequently saving costs. A second study was published by the same group,36 directly comparing the BMDCs þ PRP þ scaffold procedure vs open and arthroscopic ACI. Eightyone patients (25 BMDCs ‘‘1-step’’ technique, 10 open ACIs, and 46 arthroscopic ACIs) were included in the study. The clinical results of the 81 patients at 3-year follow-up were compared and showed a significant improvement for each treatment without any intergroup difference, thus confirming the affordability of this single-step technique. The same technique was tested for osteochondral defects of the femoral condyles in 20 patients.37 The outcome significantly improved in each of the scores used at the 24-month evaluation, and MRI findings were also positive. Interesting findings were a slower recovery but a similar outcome in cases with combined surgery with respect to patients who underwent the cartilage procedure alone; moreover, the hyperintense signal of the repairing tissue in MRI appeared to be related to a poorer clinical outcome. Generally, all clinical studies present a weak methodology owing to the presence of several combined confounding treatments that limit the assessment of PRP’s contribution, or the lack of a control group or both, and therefore they can only suggest a possible role of PRP as an augmentation procedure.

Discussion The idea of promoting tissue regeneration is one of the leading concepts in current orthopaedic practice, also considering that practitioners often face degenerative lesions of tissues, such as tendon, muscle, and cartilage, with low healing potential. For this purpose, several novel strategies have been developed over time, and both scaffold technology and GFs are suitable ways of achieving this goal. An extensive application of such methods has been documented in several in vitro and in vivo studies, which has led, in some cases, to the clinical use of these products. In particular, PRP has been tested both during surgical procedures as a biological augmentation32-37 and as a conservative injective management for cartilage pathology,38 whereas scaffolds (including some particular PRP preparations) are peculiarly used in a surgical setting. Because the rationale is almost the same, and is based on the fact that the features of scaffolds and PRP might theoretically be complementary, it was to be expected that PRP-augmented scaffolds would ultimately be studied. As for the preclinical evidence, results were quite encouraging because in most of the trials analyzed, especially the ones

113 dealing with animal models, PRP administration seemed to improve the quality of the repair tissue without affecting the mechanical properties of the scaffold. However, there was also a trial revealing that PRP might be detrimental when added to a collagen-hydroxyapatite scaffold, leading to poorer histologic and immunohistochemical results when compared with the ‘‘bare’’ scaffold. That was an interesting finding because it raises the most important issue concerning biological and bioengineered strategies for cartilage repair, which is the extreme variability of products and techniques used. Every scaffold, in fact, possesses unique chemical and biomechanical properties affecting its osteoconductive and osteoinductive potential, and therefore the possibility to achieve a good interaction with platelet concentrates and a good quality repair tissue. In light of this, each biomaterial might respond differently to GF supplementation and a further variable has to be considered, that is, the different techniques used to bond PRP to scaffolds. The particular strategy used might determine different patterns of GF release and biochemical modifications able to affect the biological results. Moreover, it has to be remembered that there are several PRP formulations, differing in preparation methods, cell content, activation, and many others aspects.39 Extensive research is being conducted currently to understand better the basic biology behind PRP because, although some GFs might be fundamental for cartilage healing, other GFs contained in PRP are under investigation for impairing tissue regeneration.40 Another confounding factor is the role of the cells that are often added to scaffolds. Generally, cultured chondrocytes are employed but so are mesenchymal stem cells derived from bone marrow or adipose tissue. The response of these cells is different after GF stimulation31 and their biological properties are largely unknown. Finally, we have to consider the different animal models where these augmented scaffolds have been tested, because different behavior patterns might be observed with the same scaffold applied in different animals. The large number of variables involved is crucial and explains the difficulty in understanding the contribution of plateletderived GFs to the scaffold-mediated regenerative processes. In consideration of the current understanding of the efficacy and feasibility of applying PRP-augmented scaffolds to clinical practice, it is still not possible to assess conclusive findings. In fact, looking at the available trials up to now, it is very difficult to identify to what extent PRP might contribute to determining the clinical outcome with respect to the surgical treatment performed with the scaffold alone. Comparative studies aimed at assessing the specific role of PRP are needed. Furthermore, in most cases, PRP is administered together with other biological augmentation methods such as mesenchymal stem cells.35-37 Therefore, it is even more difficult to determine the contribution of PRP itself. The studies available are limited in terms of clear-cut evidence, as they are case series treating disparate conditions in biomechanically very different joints (knee and ankle). In addition, maximum follow-up evaluation is 24 months, so further trials are needed to determine the persistence of the good clinical outcome of these particular procedures.

114 In the near future, both scaffolds, PRP and cells, will be increasingly used in clinical practice, alone or in combination, and we have to expect that other biological approaches would also be proposed. The risk is that the ‘‘augmentation theory’’ would invade clinical practice without a proper knowledge of the biological contribution of the single components and the entire bioengineered device. The temptation to add more biological products to treat cartilage pathology, despite the good clinical outcome reported, might, in the end, impair a better understanding of potentials and limits of new biological approaches. Properly designed, robust scientific studies should focus on demonstrating the actual role of PRP as an augmentation procedure, and even then, research should be performed to define the best clinical indications, thus developing a profile of patients and lesions that would most benefit from these biological strategies.

Conclusions Considering preclinical and clinical studies, the current literature shows encouraging results. However, reports on the use of PRP-augmented scaffolds are still preliminary, with a low scientific level. Moreover, the simultaneous presence of different treatments limits the interpretation of the role of each one separately, thus making it less clear to understand the actual role of PRP in enhancing scaffold properties. Clinicians should be aware that current clinical evidence does not allow PRP augmentation to be recommended for scaffold-based cartilage repair, and the indiscriminate use of biological strategies should be avoided, as it would not be possible to clarify important aspects that are the biological foundations of this kind of approach. ‘‘The more, the better’’ is not always true, and physicians should be aware of this and proceed cautiously when applying combinations of new biological treatments.

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37.

38.

39.

40.

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