Stem cell- and growth factor-based regenerative therapies for avascular necrosis of the femoral head

Avascular necrosis (AVN) of the femoral head is a debilitating disease of multifactorial genesis, predominately affects young patients, and often leads to the development of secondary osteoarthritis. The evolving field of regenerative medicine offers promising treatment strategies using cells, biomaterial scaffolds, and bioactive factors, which might improve clinical outcome. Early stages of AVN with preserved structural integrity of the subchondral plate are accessible to retrograde surgical procedures, such as core decompression to reduce the intraosseous pressure and to induce bone remodeling. The additive application of concentrated bone marrow aspirates, ex vivo expanded mesenchymal stem cells, and osteogenic or angiogenic growth factors (or both) holds great potential to improve bone regeneration. In contrast, advanced stages of AVN with collapsed subchondral bone require an osteochondral reconstruction to preserve the physiological joint function. Analogously to strategies for osteochondral reconstruction in the knee, anterograde surgical techniques, such as osteochondral transplantation (mosaicplasty), matrix-based autologous chondrocyte implantation, or the use of acellular scaffolds alone, might preserve joint function and reduce the need for hip replacement. This review summarizes recent experimental accomplishments and initial clinical findings in the field of regenerative medicine which apply cells, growth factors, and matrices to address the clinical problem of AVN.


Introduction
Osteonecrosis or avascular necrosis (AVN) is defi ned as a pathologic process that results from a critical reduction of blood supply to the bone and elevated intraosseous pressure. Although the pathogenic process is poorly understood, osteonecrosis is the fi nal common pathway of traumatic and non-traumatic insults and compromises the already precarious circulation of the femoral head. Femoral head ischemia subsequently leads to the death of bone marrow and osteocytes and usually results in the collapse of the necrotic segment [1]. AVN is a debilitating disease that usually leads to osteoarthritis of the hip joint in young adults. Th e disease prevalence is unknown, but estimates indicate that 10,000 to 20,000 new cases are diagnosed in the US each year. Furthermore, it is estimated that 5% to 18% of the more than 500,000 total hip arthroplasties performed annually are related to advanced AVN [2]. Joint-preserving techniques such as core decom pression, avascular or vascularized bone graft ing, and various femoral osteotomies are most promising in early stages of AVN -Association Research Circulation Osseous (ARCO) stage I and II -with preserved structural integrity of the subchondral plate [2]. Th e structural collapse of the osteonecrotic segment (ARCO stage III and IV) indicates the progressive course of the disease, and total hip replacement is inevitable in most cases in the long term [1,3].

Cell-based strategies
To enhance osseous regeneration, the application of osteogenic or angiogenic precursor cells (or both) in combination with or without supporting growth factors is an appealing possibility. Among the various cell types, adult tissue-derived, multipotent mesenchymal stem cells (MSCs) represent a highly promising candidate for cellbased therapeutic approaches [4]. Th ese somatic adult stem cells can be detected in specifi c tissue compartments of the human body and are considered to play a critical role in maintaining the integrity of diff erent tissues such as skin, bone, and blood [5]. MSCs maintain the ability of mitotic multiplication without signifi cant loss of their specifi c biomolecular characteristics over extensive population expansion and are capable of diff eren tiating into multiple mesenchymal phenotypes, including osteoblasts, chondrocytes, and adipocytes [5]. MSCs have been shown to enhance tissue regeneration after transplantation in an experimental AVN model in dogs [6]. However, whether the observed eff ects resulted from the osteogenic diff erentiation of transplanted cells or are mediated via their trophic activities remains unclear [6,7].
Proper vascularization is essential for the viability and function of the majority of tissues in the body. Without suffi cient blood supply, cells suff er from hypoxia, lack of nutrients, and the accumulation of waste products, and biochemical signaling mechanisms are disrupted, aff ecting tissue homeostasis and preventing tissue regeneration. Endothelial progenitor cells (EPCs) are spindleshaped cells capable of diff erentiation into a mature endo thelial phenotype. Th ese cells can be isolated from bone marrow and peripheral blood [8,9]. Th eir role in angiogenesis and neovascularization has been studied extensively, and positive eff ects on blood vessel formation after transplantation have been reported [10]. So far, however, these cells have not been applied clinically for the treatment of AVN.
Interestingly, Feng and colleagues [9] recently reported signifi cantly decreased numbers of circulating EPCs and colony-forming units (CFUs) in patients with diagnosed AVN in comparison with a healthy control group. Furthermore, EPCs of patients with AVN exhibited impaired migratory capacities and increased cellular senescence, resulting in reduced angiogenesis in vitro [9].

Application of concentrated bone marrow aspirates
Hernigou and colleagues [11][12][13] pioneered the clinical application of a cell-based strategy for the treatment of AVN, percutaneously injecting an autologous bone marrow concentrate into the necrotic area of femoral heads. Th e strategy is driven by the hypothesis that injected bone marrow cells can repopulate the trabecular bone structure and subsequently revitalize and remodel the necrotic bone. For this technique, a bone marrow aspirate is harvested from the iliac crest and the mononuclear cell fraction is isolated. After core decompression, the cell suspension is injected into the necrotic area [11][12][13]. Th ese authors reported on the postoperative outcome in 189 cases (116 patients) treated with the injection of autologous bone marrow concentrate [11]. Over the course of the 5-to 10-year follow-up, nine out of 145 patients with early-stage AVN (Steinberg stage I or II) and 25 out of 45 patients with later-stage AVN (Steinberg stage III or IV) required total hip replacement. However, since no control group was examined, the benefi cial eff ect of the injected bone marrow concentrate (that is, in comparison with sole core decompression) is diffi cult to prove. Gangji and Hauzeur [14] conducted a controlled prospective study comparing the postoperative outcome of core decompression with core decompression followed by the injection of a bone marrow concentrate in early-stage AVN (ARCO stage I or II). Th e mean follow-up was 2 years, and fi ve out of eight hip joints in the control group showed radiographic progression. In contrast, only one out of 10 hips treated with bone marrow cells progressed to a more severe stage with collapse of the subchondral bone [14]. Recently, Gangji and colleagues [15] reported on the 5-year follow-up of 19 patients (24 hip joints) who received either core decompression alone or core decom pression with the additional application of bone marrow concentrate because of early-stage AVN. In this prospective, doubleblinded trial, eight out of 11 hip joints in the core decompression group and only three out of 13 joints in the bone marrow concentrate group revealed a disease progression with structural disintegra tion of the subchondral bone [15]. Despite the promising results, the clinical value of these studies is limited because of shortterm follow-up periods and low case numbers.

Application of ex vivo expanded autologous bone marrowderived stem cells
Th e ex vivo amplifi cation and further administration of MSCs, in contrast to bone marrow cell concentrates, are controlled by regulatory authorities, namely the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) [16]. In most cases, the selec tion of MSCs is achieved by cell adherence to tissue culture plastic after phase gradient separation (that is, Ficoll or Percoll).
Th e therapeutic potential of ex vivo amplifi ed bone marrow-derived MSCs for the treatment of corticosteroid-induced AVN in femoral condyles was investigated by Müller and colleagues [17]. In fi ve patients, bone marrow aspirates were harvested from the posterior iliac crest and MSCs were isolated and expanded for three passages. Th e MSCs were resuspended in 0.1% serum albumin-supplemented saline and transplanted into the necrotic area. After a mean follow-up of 16 months, none of the patients showed signs of disease progression. Owing to the lack of a control group, whether this eff ect can be attributed to the injected cells is unclear.
Apart from cell delivery in suspension, a variety of matrices, such as ceramics, collagen sponges, hydrogels, and biodegradable polymers, have been used for cell delivery [4]. Core decompression off ers the opportunity to deliver such cell-laden biomaterials to the necrotic area. Concurrently with tissue neoformation, scaff olds ideally undergo degradation processes resulting in biocompatible, metabolisable, and excretable by-products.
Specifi cally, for the treatment of AVN, autologous or allogenic bone grafts (demineralized bone matrix, or DBM) as well as synthetic biomaterials (that is, betatricalcium phosphate, or β-TCP) have been suggested as suitable carriers for cell-based strategies [3,4].
Kawate and colleagues [18] reported the treatment of three patients with advanced stages of cortisone-induced AVN (Steinberg stage III or IV) with a vascularized fi bular graft combined with a synthetic β-TCP ceramic and bone marrow-derived MSCs. Four weeks prior to elective core decompression, 15 mL of bone marrow aspirate was taken from the iliac crest. MSCs were isolated and expanded in autologous serum. After 10 days, MSCs were seeded onto the β-TCP granula and cultured for 2 weeks. After core decompression, the defect was fi lled with the MSC-seeded β-TCP granula and a vascularized fi bular graft was transplanted. During the 34-month follow-up, no progression of the AVN was reported [18]. Given the advanced stages treated and the cortisone-related genesis of the necrosis (both of which have been shown to lead to a decreased therapeutic response in general [3,19]), these results are promising. However, only a small number of patients were treated and a control group was not included.
A similar approach to treat AVN with autologous stromal cell-seeded β-TCP granula was presented by our group [20]. Th e applied cells consisted of a mixture of MSCs and endothelial and hematopoietic progenitor cells (Tissue Repair Cells, or TRC; Aastrom Biosciences, Inc., Ann Arbor, MI, USA).
Briefl y, bone marrow aspirates (100 mL) were harvested bilaterally from the posterior iliac crest. Th e aspirates were expanded for 12 days in autologous serum-supplemented medium by means of a good manufacturing practice (GMP) system (Aastrom Biosciences, Inc.). Fluores cence-activated cell sorting analysis of the expand ed cells showed a signifi cantly higher number of CFU-fi broblasts and cells expressing surface markers characteristic of MSCs (that is, CD90) and hematopoietic (that is, CD133) and endothelial (that is, CD90) progenitor cells when compared with the mononuclear fraction of native bone marrow aspirates, indicating an enrichment of these cell types [20]. Early AVN (ARCO stage II) was diagnosed in all treated patients. After surgical exposure of the proximal femur, a bone plug was removed by using a hollow diamond drill. For the treatment of small AVN lesions (ARCO stage IIA), a Kwire was placed centrally into the necrotic area and was used as a guide for a cannulated 10-mm drill bit. For the treatment of more extensive AVN (ARCO stage IIB and IIC), two drill holes were used ( Figure 1A-D) [20]. Subsequently, β-TCP granula (Vitoss; Orthovita Inc., Malvern, PA, USA) were placed in a custom-made plastic tray and the TRC suspension was homogeneously seeded onto the granula (Figure 2A). For ease of handling, autologous plasma obtained during surgery was used to achieve physical bonding between the MSC-seeded β-TCP granula ( Figure 2B). Th e tissue-engineered construct consisting of cells, β-TCP granula, and autologous plasma was administered to the necrotic area through a plastic funnel (Orthovita Inc.) ( Figure 2C,D), and the drill hole was sealed with the preserved bony cylinder.
At present, a total of four patients have been treated by using the described technique without disease progression ( Figure 3A-D) at the 24-month follow-up. A European Union-funded (FP-7) clinical phase I and II study is in preparation to evaluate the benefi cial eff ects of bone marrow-derived progenitor cell application compared with standard core decompression in the treatment of early-stage AVN [21].

Application of allogenic bone marrow-derived stem cells
Th e use of allogenic instead of autologous MSCs for the treatment of AVN appears attractive because of logistic and economic advantages given that these cells might be available as an 'off the shelf ' product. However, allogenic MSCs harbor the danger of disease transmission and immunological rejection [22], as observed in organ transplantation. Th erefore, a peculiar risk-benefi t analysis of allogenic MSC-based strategies in large populations of patients has to be addressed, especially when aiming for non-life-threatening diseases such as AVN. Because alternative treatment options are lacking, allogenic MSC transplantation appears to be more suitable in severe disorders such as osteogenesis imperfecta. Cell engraftment and accelerated growth velocity have been shown in children with severe osteogenesis imperfecta after systemic administration of isolated allogenic bone marrow-derived MSCs [23,24].

Growth factor-based strategies
A variety of growth factors produced by osteogenic cells, platelets, and infl ammatory cells -including bone morpho genetic proteins (BMPs) [25][26][27], insulin-like growth factor-1 and -2, transforming growth factor-β1 (TGF-β1), platelet-derived growth factor, and fi broblast growth factor-2 -are functionally involved in bone healing. Th e bone matrix serves as a reservoir for these growth factors, which are activated during matrix resorption by matrix metalloproteases. Additionally, the acidic environment that develops during the infl ammatory process leads to activation of latent growth factors, which assist in chemo-attraction, migration, proliferation, and diff erentiation of MSCs into osteoblasts or chondroblasts [25][26][27]. All of these functions are driven by complex interactions among growth factors and other cytokines and are infl uenced by multiple regulatory factors. Th us, the use of osteogenic growth or diff erentiation factors for joint-preserving treatment of AVN is also a potentially promising approach [28].

Osteogenic growth factors Bone morphogenetic proteins
Healing of osseous tissue is regulated mainly by members of the TGF-β superfamily. Specifi cally, BMPs act to induce the formation of both bone and cartilage by stimulating mesenchymal progenitor cells. However, only a subset of BMPs, notably BMP-2, -4, -7, and -9, have been shown to exhibit osteoinductive activity in de novo bone formation [25][26][27][28]. Lieberman and colleagues [29] treated 15 patients (17 hip joints at Ficat stage II or III) after core decompression with allogenic, antigen-extracted, autolyzed fi bula grafts, combined with 50 mg of recombinant human BMP-2 and non-collagenous protein.
During the mean follow-up of 53 months, three patients (one Ficat stage II and two Ficat stage III) required total hip replacement because of an aggravation of clinical symptoms corresponding with radiographic progression [29].
Mont and colleagues [30] aug mented bony defects result ing from AVN with a triphasic bone substitute consisting of DBM, processed allograft bone chips, and a thermoplastic carrier plus the addition of BMP-7 in 19 patients (21 hip joints). Eighty-six percent of the treated hips were assessed as positive outcomes -Harris Hip  Score (HHS) of at least 80 -at the fi nal follow-up (mean follow-up of 48 months) [30]. Th e same group conducted a retrospective cohort study in 33 patients (39 hip joints:  22 at Ficat stage II and 17 at Ficat stage III) implanted with autologous, non-vascularized bone grafts loaded with BMP-7. At the fi nal follow-up (mean of 36 months), only four out of 22 Ficat stage II hips and 11 out of 17 Ficat stage III hips had undergone hip arthroplasty. At present, whether the administration of BMPs results in better clinical out comes remains unclear as the literature lacks controlled randomized studies that include appropriate numbers of patients and control groups [31].
In another study, Mont and colleagues [32] created an experimental bony defect at the antero-lateral aspect of the femoral head in canines. Th e defect was augmented with an autologous bone graft from the iliac crest in combination with 250 mg of BMP-7 per gram of autograft. In the control group, the defect remained untreated. Four months after surgery, all control animals showed a collapse of the femoral head with degeneration of the hyaline cartilage. Autograft augmentation with or without BMP-7 preserved the subchondral bone and the production of a surface layer of hyaline cartilage. Indepen dently of BMP supplementation, radiographs revealed good to excellent osseous remodeling when autograft augmentation was performed [32].
Th e healing potential of BMP-2-and BMP-14-loaded collagen scaff olds was investigated by Simank and colleagues [33] in a sheep model. Necrosis of the femoral head was created by direct ethanol injection. After core decompression, BMP-laden scaff olds were transplanted and compared with non-loaded scaff olds. Th ree months after surgery, both growth factor-treated groups revealed better bone regeneration histomorphologically [33]. When the same animal model was used, BMP-14-loaded, hydroxyapatite-coated collagen scaff olds (Healos; DePuy, Warsaw, IN, USA) signifi cantly improved bony remodeling of the necrotic area. In comparison, when the scaff old was applied without BMP-14, only partial defect fi lling with mainly fi brous tissue and persistence of the necrosis was seen after 12 weeks [34].
To achieve long-term availability of osteoinductive or angiogenic growth factors in a defect, it is also possible to deliver gene-modifi ed cells synthesizing and secreting the desired protein. Genetic engineering strategies focusing on osteoinductive factors have emerged as effi cient approaches to enhance bone formation. Two modalities are generally used: (a) direct in vivo delivery of gene constructs and (b) ex vivo transduction and subsequent transplantation of cells expressing the osteoinductive factor [4]. Th e choice of the gene delivery method depends on several factors, including the particular gene of interest, indication targeted, desired duration of gene expression, and nature of the delivery vector.
Tang and colleagues [35] induced bilateral AVN in goats by ligation of the lateral and medial circumfl exing arteries and additional delivery of liquid nitrogen to the femoral head. Th e authors investigated the infl uence of MSCs, transduced with either BMP-2 or β-galactosidase and seeded onto β-TCP scaff olds on AVN consolidation. Th ree weeks after core decompression, the tissueengineered constructs were transplanted. Whereas control animals (core decompression only) showed AVN progression with structural disintegration and collapse of the subchondral bone 4 months after treatment, no signs of such progression were observed in the cell-treated groups [35]. In the case of BMP-2 transduction, superior bone regeneration in the defects was observed when compared with the β-galactosidase group, as were signifi cantly higher amounts of new bone and higher maximum compressive strength and bone density [35].

Angiogenic growth factors Vascular endothelial growth factor
Vascular endothelial growth factor (VEGF) regulates numerous cellular events associated with angiogenesis and vasculogenesis, such as tissue remodeling, during embryonic development and in adults [36]. Yang and colleagues [37] delivered plasmids encoding VEGF immobi lized on a collagen carrier into the necrotic area of the femoral head in rabbits. Signifi cantly higher bone formation was reported 8 weeks after treatment in comparison with control animals that received a collagen carrier without VEGF plasmids. Th e successful in vivo transfection of local cells by the VEGF plasmids and expression of VEGF after surgical delivery, however, were shown up to 2 weeks only [37].

Other growth factors Granulocyte colony-stimulating factor and stem cell factor
Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein, growth factor, and cytokine with two isoforms, both of which stimulate the proliferation of bone marrow stromal cells and enhance their availability in the circulating blood volume [38]. Stem cell factor (SCF) is a cytokine that plays a critical role in regulating the diff erentiation of hematopoietic stem cells. After intramuscular application of G-CSF and SCF, Wu and colleagues [39] observed increased blood vessel formation (3.3-fold), a higher blood vessel density (2.6-fold), and increased bone formation in the necrotic area of steroid-induced AVN of rabbits in comparison with a non-treated control group. However, whether direct angiogenic/osteogenic diff erentiation of the mobi lized progenitor cells accounted for the increased vascularization and bone formation or whether an increased trophic activity of these cells activated endogenous progenitor cells at the defect site was unclear [39].

Hepatocyte growth factor
Hepatocyte growth factor (HGF) is a multifunctional cyto kine that regulates cell growth, cell motility, and morphogenesis and exhibits synergistic eff ects with VEGF [40]. Wen and colleagues [41] used a hormoneinduced AVN rabbit model to compare the therapeutic eff ect of core decompression alone versus the additional augmentation of the necrotic area with HGF-transduced MSCs administered in fi brin glue. De novo bone formation with regular trabecular structure and enhanced vascularization of the necrotic bone was observed only in animals treated with HGF-transduced MSCs as shown by magnetic resonance imaging and computed tomography imaging and immunohistochemistry [41].

Anterograde osteochondral reconstruction in advanced stages of avascular necrosis
With the progression of untreated AVN (ARCO stage III and IV), structural disintegration of the subchondral bone occurs and leads to the collapse of the femoral head. A substantial degeneration of the hyaline cartilage has to be assumed at this stage, and retrograde strategies, such as core decompression and autologous bone grafting, off er only very limited prospects [42]. In most of these cases, total joint replacement remains the only treatment option [43]. Th e potential of regenerative, joint-preser ving techniques, such as osteochondral trans plantation (auto grafts or allografts), bone grafting followed by autologous chondrocyte implantation (ACI), or acellular matrix implantation, has not been exploited suffi ciently so far. Th ese techniques include a surgical dislocation of the hip to anterogradely access the osteochondral defect, which is more invasive and surgically demanding [42,44].

Osteochondral transplantation (mosaicplasty)
Meyers and colleagues [45,46] trans planted osteochon dral autografts or allografts in 21 patients with pro gressed stages of AVN (Ficat stage II to IV) after hip dislocation and debridement of the osteo chondral defect. Th e treatment was reported to be successful in 71% to 80% of all cases (follow-up for 1.5 to 5 years) but in only 50% of the patients with steroid-induced AVN [45,46]. Sotereanos and colleagues [47] treated a 36-year-old patient after ineff ective core decom pression and vas cular ized fi bula graft augmentation. Th ree osteochondral cylinders were transferred from a non-weight-bearing area at the inferiorlateral femoral head into the defect following bone grafting. After 5 years, the HHS was 96, the patient was pain-free, and the range of motion was not limited [47].
Rittmeister and colleagues [48] performed an autologous osteochondral transfer (one to three grafts) in fi ve patients with advanced AVN. After a mean follow-up of 57 months, the therapy failed in four patients and total hip arthroplasty was required. Only one patient was treated successfully, and hip function showed no limitations 31 months after surgery [48].

Autologous chondrocyte implantation
ACI has been proven to be a suitable technology for the treatment of full-thickness cartilage defects in the knee and ankle joint. Its therapeutic potential in the hip has not been addressed suffi ciently so far. Only one case report on ACI at the femoral head has been published so far. Akimau and colleagues [49] treated a 31-year-old man with a diagnosed post-traumatic necrosis of the femoral head by using a modifi ed fi rst-generation ACI approach. Healthy hyaline cartilage (240 mg) was harvested arthroscopically from the ipsilateral knee joint, and chondrocytes were isolated and expanded in monolayer culture over a period of 3 weeks. In a second surgery, the femoral head was dislocated and exposed. After removal of the degenerated cartilage and necrotic bone, the defect was covered with a collagen type I membrane (Chondro-Gide; Geistlich, Wolhusen, Swit zer land), and 6 × 10 6 chondrocytes were injected underneath the membrane. Th e HHS increased from 45 preopera tively to 76 at 12 and 18 months after surgery. Arthro scopic biopsy revealed a 2 mm-thick repair tissue consist ing of fi brous and, to a lesser extent, hyaline-like cartilage [49].

Acellular matrix implantation
Matrix-based ACI has been shown to be a clinically successful method for the treatment of full-thickness cartilage defects by using a variety of scaff old materials [4,50,51]. In ACI, a previous cartilage biopsy harvest followed by ex vivo cell processing, which is timeconsuming and costly, is required. To circumvent these barriers, an innovative one-step procedure combining micro fractur ing [52] of the subchondral bone and coverage of the cartilage defect by using an acellular scaff old (Autolo gous Matrix-Induced Chondrogenesis) was developed [53]. We recently adopted this basic principle for the treatment of an advanced stage of AVN in the hip (ARCO stage III) as shown in Figure 4. Th e hip joint was dis located after a trochanteric fl ip osteotomy was performed [44] and the femoral head was exposed ( Figure 4). Th e osteochondral defect was debrided, the sclerotic bony ground was repeatedly penetrated by using a small drill bit, and the bony defect was augmented with autologous cancellous bone. Subsequently, the cartilage defect was replenished by using an acellular collagen type I hydrogel (CaReS-OneStep; Arthro Kinetics, Krems, Austria) [54]. So far, three patients have been treated with this technique with a follow-up of 9 months.

Conclusions
Core decompression is the gold standard technique for the treatment of early-stage AVN of the femoral head [55][56][57] and reveals superior clinical outcome in comparison with non-operative treatment regimens [58,59]. Th e variable and unpredictable clinical outcome led to the development of complex surgical techniques, such as the transplantation of non-vascularized/vascularized autologous bone grafts, aiming [31,60] for a more reliable bone regeneration and preservation of a physiological joint function. Extensive research activities over the last decade explored the potential of mesenchymal progenitor cells [4][5][6][7] and growth factors [4,5,25,27,28,35,37,54], aiming for an autologous ex vivo or in vivo regeneration of a variety of musculoskeletal tissues.
Early promising clinical data on the application of concentrated bone marrow cells, isolated bone marrowderived progenitor cells, and growth factors for the treatment of AVN have been reported [3,[11][12][13][14][15]31,32]. However, so far, both cell-and growth factor-based strategies undertaken lacked randomized clinical trials to suffi ciently validate their effi ciency in comparison with conventional therapies. Whether and to what extent the observed bone regeneration resulted from the applied cells or growth factors remain uncertain.
In addition, ex vivo processed/expanded cells are classifi ed as advanced therapy medicinal products (ATMPs) by the competent international and national regulatory agencies (for example, the FDA and the EMA). ATMPs have to fulfi ll specifi c quality and safety criteria that rely on standardized GMP conditions for cell processing, the performance of adequate preclinical animal models for the targeted disease, and controlled clinical phase I/II trials [16]. Th is increases time-and cost-consuming barriers on the way to FDA/EMA approval on the one hand but provides the opportunity to investigate the potential of cell-based strategies in musculoskeletal diseases guided by a controlled regulatory framework on the other hand. Also, owing to unresolved safety concerns, the clinical translation of gene therapy approaches for the treatment of AVN is currently not feasible at all.
Th e treatment of osteochondral defects in advanced stages of AVN, characterized by a collapse of the subchondral bone, remains an unresolved burden in orthopedic surgery [1][2][3]. As a result, the predominantly young patients usually require total hip arthroplasty [2,3,61]. At present, the applicability of regenerative joint-preserving techniques, such as matrix-based ACI or osteochondral transplantation, has not been adequately addressed. One reason is likely the technically demanding surgical hip dislocation required for anterograde access to the femoral head [44].
Applying tissue engineering-based techniques for joint preservation approaches could have great therapeutic potential in the restoration of femoral head integrity [4,28,54]. It is noteworthy that the few case reports recently published have shown heterogeneous results, ranging from complete therapeutic failure to full recovery with restored hip function [45][46][47][48][49]. Full evaluation of the promising therapeutic potential of these novel regenerative strategies for AVN will require rigorous randomized controlled trials that address stage-dependent treatment of the disease.

Competing interests
The authors declare that they have no competing interests.

Author contributions
LR helped to prepare the outline, write the initial draft of the manuscript, and develop the acellular matrix implantation surgical technique presented here. LE, SR, and JCR helped to prepare the outline and write the initial draft of the manuscript. UN and MR helped to edit and revise both the outline and the draft of the manuscript, provided additional references and insights, and helped to develop the acellular matrix implantation surgical technique presented here. FJ, HW, OP, and RST helped to edit and revise both the outline