A Method for the Immunohistochemical Identification and Localization of Osterix in Periosteum-Wrapped Constructs for Tissue Engineering of Bone

Abstract

A novel immunohistochemistry (IHC) approach has been developed to label and localize osterix, a bone-specific transcription factor, within formalin-fixed, paraffin-embedded, tissue-engineered constructs uniquely containing synthetic polymers and human periosteal tissue. Generally, such specimens consisting in part of polymeric materials and mineral are particularly difficult for IHC identification of proteins. Samples here were fabricated from human periosteum, electrospun poly-l-lactic acid (PLLA) nanofibers, and polycaprolactone/poly-l-lactic acid (PCL/PLLA, 75/25) scaffolds and harvested following 10 weeks of implantation in athymic mice. Heat-induced and protease-induced epitope retrieval methods from selected existing protocols were examined to identify osterix. All such protease-induced techniques were unsuccessful. Heat-induced retrieval gave positive results for osterix immunohistochemical staining in sodium citrate/EDTA/Tween 20 with high heat (120C) and pressure (~30 psi) for 10 min, but the heat and pressure levels resulted in tissue damage and section delamination from slides that limited protocol effectiveness. Heat-induced epitope retrieval led to other osterix-positive staining results achieved with minimal impact on structural integrity of the tissue and polymers in sodium citrate/EDTA/Tween 20 buffer at 60C and normal pressure (14.5 psi) for 72 hr. The latter approach identified osterix-positive cells by IHC within periosteal tissue, layers of electrospun PLLA nanofibers, and underlying PCL/PLLA scaffolds of the tissue-engineered constructs.

Introduction

This laboratory has been concerned for some time with finding solutions for the treatment and healing of skeletal defects in individuals suffering congenital abnormalities, injury, trauma, disease, and aging. Tissue engineering offers the possibility of augmenting, repairing, or replacing bone and cartilage affected in these instances. In this context, recent work has led to the development of tissue-engineered constructs consisting of human donor periosteum, electrospun poly-l-lactic acid (PLLA) nanofibers, and polycaprolactone/poly-l-lactic acid (PCL/PLLA, 75/25) scaffolds.¹ Periosteum is a very thin membrane overlying numerous bones in the bodies of vertebrates, and it contains progenitor cells ultimately giving rise to and maintaining the committed bone cells (osteoblasts) and their mineralized extracellular matrix characteristic of this tissue.² Such constructs are novel in the sense that their PCL/PLLA scaffold core may be designed in any shape and coated by electrospinning over all its surfaces.¹ Periosteum is then wrapped about the electrospun PLLA. Ultimately, these constructs could be applied to heal impaired bone.

As an initial step in testing the viability and investigating other characteristics of its constituent periosteal cells, the constructs just described are typically implanted in athymic (nu/nu, nude) mice for various periods to allow the tissues to grow, and the elaborated specimens are then harvested for extensive analysis. The mice are specially bred and, without a thymus gland, will not reject foreign tissue, human periosteum in this case. The mice then serve as small bioreactors and surrogates to a human individual who would otherwise grow his or her own replacement tissue-engineered periosteum and bone.³

Following construct implantation in the mice, viable donor periosteum will have synthesized and secreted osterix, a critical transcription factor expressed by progenitor cells undergoing differentiation to osteoblasts which secrete the extracellular matrix necessary for bone formation. Identification and localization of osterix in periosteum were chosen for examination in the present study because the pathway for bone regeneration by an osteoblastic lineage would be confirmed and thus establish the validity of using periosteum in this tissue-engineering approach.

Immunohistochemistry (IHC) is an excellent method that provides a means of identifying and localizing a protein such as osterix within a tissue through microscopic visualization, and the basic technique is used in many fields of biological research.^4,5 With IHC, protein identification and localization occur using primary antibodies as markers conjugated either to secondary molecules eliciting color under visible or fluorescent light microscopy or to functionalized gold particles and detected by light or electron microscopy.^4–11 IHC has versatility in its application in that it has a wide spectrum of approaches to recover, tag, and visualize any number of epitopes, a sequence or sequences of amino acids that comprise the protein of interest and that are recognized and bound by the specific marker. For the most common proteins in biological tissues, there are many standardized IHC protocols already developed. However, protocols for less prominent proteins are unavailable or may have relevance to specific tissues that are not valid for all applications. In such cases, a great deal of time and effort are often expended designing and refining new methods to ensure that IHC results may be achieved successfully.

In the present studies in this laboratory, implanted and harvested tissue-engineered constructs were routinely fixed in formalin and embedded in paraffin. Biological material processed in this manner may present some difficulty for IHC processing, compounded by the presence of polymeric materials serving as a scaffold for cell and extracellular matrix support and development. Indeed, while IHC has been applied successfully for osterix detection in mineralizing tissues,^12,13 the same method directed to obtain preliminary results for osterix identification and localization in the polymer- and mineral-containing constructs here was inconsistent at best. Improvement in IHC was consequently required to confirm osterix in the specimens and the potential validity for using these periosteum-enclosed constructs in the tissue engineering of bone.

A critical factor in IHC tagging or labeling a constituent protein in such formalin-fixed, paraffin-embedded tissues is the epitope retrieval process. During tissue fixation and embedding for microscopy, many proteins have their three-dimensional structure altered to the point where an antibody or marker fails to recognize or cannot locate the epitope to which it should bind.^14,15 Numerous methods have been designed to recover protein structure from a fixed and embedded tissue, therefore, and these methods are generally separated into two classes: heat-induced epitope retrieval (HIER)^16–18 or protease-induced epitope retrieval (PIER).^19–21 Both methods function as their titles suggest. HIER relies on elevated temperature to unfold a protein and expose its epitope for antibody recognition of the target motif. PIER uses a class of enzymes, proteases, which cleave proteins and accomplish the same goal as HIER.

A challenge in the application of either HIER or PIER lies in determining the optimal approach from the enormously wide array of potential conditions that may be applied for epitope retrieval. In this regard, HIER conditions include buffer composition and pH in addition to temperature variability. Achieving various temperature targets may be accomplished in multiple ways, incorporating the use of vegetable steamers, water baths, microwaves, autoclaves, or pressure cookers, each used with varying degrees of success.^16–18 Microwaving and pressure-cooking methods comprise the majority of HIER protocols for two reasons, control and time. Both can be regulated tightly for temperature and only require minutes to unmask an epitope. However, high temperature invariably causes some tissue damage and creates issues hindering the identification, localization, and quantitation of an intended target.^10,22 Should time be less a factor, methods using a water bath produce similar results under “gentler” conditions.²² Tissue structure is preserved, and the location and quantity of the target protein are easier to examine.¹⁰

PIER is different from HIER because enzymatic activity requires lower temperatures and more tightly controlled solution parameters than those with heat-induced methods. Problems may arise, however, when a tissue is digested extensively with a protease. Tissue structure is altered, and the target protein is cleaved to the point where antibody recognition fails in a worst-case scenario. There are numerous enzymes such as pronase, hyaluronidase, pepsin, and collagenases, for example, available as potential candidates for PIER application.^19–21

PIER effectiveness depends on the selected enzyme being able to remove cross-linked proteins that block an epitope or cleave parts of the protein to expose the epitope to the primary antibody. Some proteins require PIER when physical heating methods are insufficient or impractical.¹⁵

In the work here, retrieval methods were complicated by several additional parameters beyond fixation with formalin. As mentioned above, the specimens are composites of tissue that are regenerating on a polymeric scaffold material that is providing support to cells and extracellular matrix. The tissue also has the potential to mineralize over time and form apatite crystals within the constructs, and the scaffold may be electrically charged synthetic polymers such as PLLA and PCL/PLLA used in the present experiments. These scaffolds combined with attached proliferating osteoblasts required longer fixation times for preservation of the constructs during IHC processing. Longer fixation times are not uncommon with mineralized tissues, have minimal effects with most antibodies, and can be adjusted according to different epitope retrieval methods.^23,24

HIER involving varying buffers and temperatures and PIER with different proteases were examined for their efficacy in revealing osterix protein for labeling while causing as little degradation or alteration as possible to the morphology of tissue samples. Despite occasional intrinsically high background staining in constructs composed in part of the charged PLLA and PCL/PLLA polymers as noted above, the HIER technique was found to be most suitable for obtaining definitive and reproducible results that identified and localized osterix in the tissue-engineered specimens of interest following 10 weeks of implantation in the host athymic mice.

Materials and Methods

Source of Tissue Specimens

Tissue samples investigated in the following IHC experiments were obtained from tissue-engineered bone specimens. Cadaveric human tissue was donated by Rush University Medical Center (Chicago, IL) through the Gift of Hope Organ & Tissue Donor Network (Itasca, IL). Following details in previous work,³ strips of human periosteal tissue (0.6 cm × 2.5 cm) were harvested from the knee of a 51-year-old female postmortem and placed, cambium-layer face down, in petri dishes containing complete cell culture medium (M199, 10% fetal bovine serum, 1% penicillin/streptomycin, and 0.2% primocin). Explant cultures were maintained in an incubator (37C, 5% CO₂) for up to 1 week with complete culture medium changes every 48 hr. After 1 week in culture, periosteal tissue strips were wrapped around polymer scaffolds composed of PCL/PLLA (75/25) and coated with thin layers (5–10 µm) of electrospun PLLA nanofibers.¹ These periosteum-wrapped constructs were implanted subcutaneously in athymic mice (Envigo, Indianapolis, IN) which were housed and maintained in accordance with regulations of the Institutional Animal Care and Use Committee at the Northeast Ohio Medical University (Rootstown, OH). After a period of 10 weeks of implantation, mice were sacrificed by CO₂ euthanasia and constructs were retrieved and fixed immediately in 10% neutral-buffered formalin (NBF). Constructs remained in formalin for 1 week. Samples were then passed through an automated tissue processor (Model ASP 300S; Leica, Buffalo Grove, IL) according to an established methodology²³ (with the minor change of using xylene [two changes, 60 min each] in place of a xylene substitute) and embedded in paraffin.

Sectioning and Initial Slide Preparation

Before sectioning, the formalin-fixed paraffin-embedded specimens of PCL/PLLA coated with electrospun PLLA nanofibers and wrapped with human donor periosteal tissue were placed overnight in a −80C freezer. They were then removed from the freezer, attached cold to a holder of a microtome (Model RM2255; Leica), and sectioned (4–7 µm thickness) using a tungsten carbide D-profile knife. Sections were next floated on a 42C water bath, mounted onto glass slides (Superfrost Excell, Thermo Scientific, Waltham, MA), and left on a warming platform to dry at 42C for a minimum of 3 hr. Slides were subsequently stored in slide boxes before histological or immunohistochemical staining. Undecalcified sections were stained with a von Kossa solution to demonstrate phosphate (both organic and inorganic phosphate as the two are indistinguishable with von Kossa staining), and alizarin red to show calcium.^25,26

In preparation for IHC, sections on slides were heated in a 60C oven and then deparaffinized through three fresh changes of xylene (10 min each; VWR Intl., Radnor, PA). They were rehydrated using 100% ethanol (1 × 10 min, 1 × 5 min) and 95% ethanol (2 × 5 min each). Individual slides were dried with cool air from a heat gun (5 amp, 93C/149C; Master Appliance Corp., Racine, WI) for a period of 20 min. The slides were rehydrated through two changes of deionized water (5 min each). Rehydrated slides were then moved to a Coplin jar and fixed in 10% NBF (VWR Intl.) overnight at 4C. The following morning, NBF was exchanged for fresh Immunocal (two changes, 1 hr each; Decal Chemical Corp., Tallman, NY) to decalcify slide-mounted sections. Slides were rinsed with deionized water (2 × 5 min each) following the second Immunocal rinse and before addition of a peroxide-blocking solution (0.3% H₂O₂/30% CH₃OH, 30 min).

Protease-Induced Epitope Retrieval

Table 1 presents results from a variety of epitope retrieval methods used to label osterix in tissue-engineered constructs containing periosteum, PLLA nanofibers, and PCL/PLLA scaffolds. PIER was attempted using type 1 collagenase (0.1% in phosphate-buffered saline [PBS]), pronase (0.1% in PBS), and a mixture of the two (0.1% each in PBS). Protease solutions were added dropwise to individual specimens and incubated at 37C in a humidified chamber for a period of 30 min for each protocol. After incubation, slides were left to cool to room temperature (~23C).

Table 1.

Osterix Epitope Retrieval Solution, Temperature, and Time Parameters.

Antigen Retrieval Solution	Temperature (C)	Time	Result
HIER
10-mM sodium citrate/2-mM EDTA/0.05% Tween 20 (pH 6.0)	60	72 hr	++
	65	1 hr	−
		24 hr	−
	95	10 min	−
	120 (*)	10 min	+
10-mM sodium citrate/0.05% SDS (pH 6.0)	65	1 hr	NA
		24 hr	NA
10-mM Tris-HCl (pH 9.0)	65	1 hr	−
0.2 M boric acid (pH 7.0)	65	44 hr	−
PIER
0.1% pronase	37	30 min	−
0.1% type 1 collagenase	37	30 min	−
0.1% pronase/0.1% type 1 collagenase	37	30 min	−

Results were categorized as unsuccessful (-), positive with tissue damage (+), or positive and lacking visible tissue damage (++). N/A for results indicates that the sections of tissue and polymer delaminated from slides completely and prevented conclusive assessment of the epitope retrieval method. For HIER, heat sources were a water bath (60-95C) or an autoclave (120C, marked with an asterisk (*)). PIER was carried out in an oven set to 37C.

Abbreviations: HIER, heat-induced epitope retrieval; PIER, protease-induced epitope retrieval.

Heat-Induced Epitope Retrieval

Buffer solution formulations are outlined in Table 1 for HIER methods. Slides were placed in staining dishes containing each buffer solution before heating. Heat sources were a water bath (60–95C) or an autoclave (120C). Autoclaved slides were subjected to increased pressure (14.5 psi above atmospheric, ~30 psi) in addition to high temperature. After heating, slides were left in the buffer to cool to room temperature (~23C) before continuing procedures.

Osterix Antibody Labeling

Following each retrieval method, slides were transferred, face up, to a humidified chamber and Background Buster (Innovex Biosciences, Richmond, CA) was added as the protein block (30 min). Protein blocking was trailed immediately with addition of primary antibody recommended for osterix detection of human origin (rabbit purified affinity polyclonal antibody, OSX (Y-21): sc-133871, dilution 1:50; Santa Cruz Biotechnology, Dallas, TX). Negative control sections (specimens receiving no primary antibody) were covered with Tween 20/Tris-buffered saline (TTBS) so the sections remained hydrated. The humidified chamber containing the slides was placed in a 4C refrigerator overnight. To validate the optimal dilution of primary antibody suggested as 1:50 by the manufacturer (Santa Cruz Biotechnology), a series of dilutions (1:50, 1:100, 1:250, and 1:500) in Bond primary antibody diluent (Leica) was used and photomicrographs of construct sections were recorded and compared. Counterpart controls using anti-rabbit IgG (horseradish peroxidase–conjugated, goat polyclonal; Bethyl Laboratories, Inc., Montgomery, TX) alone at the same working dilutions were also examined and imaged (Fig. 1).

Figure 1.

Comparison of staining in representative sections of constructs following treatment with osterix primary antibody diluted (A) 1:50, (B) 1:100, (C) 1:250, and (D) 1:500 in Bond primary antibody diluent. A representative section of one construct from among all specimen counterparts processed in the identical manner as the dilution series but with the primary antibody replaced by the anti-rabbit IgG control (E; example of the absence of staining with the anti-rabbit IgG control diluted at 1:50). Constructs consisted of human periosteum (P) wrapped about an underlying PCL/PLLA (75/25) scaffold. A layer of electrospun PLLA nanofibers was deposited on the scaffold surfaces but it is not immediately apparent in these images. Constructs were retrieved after implantation for 10 weeks in athymic mice and were decalcified in the same fashion. They were identically treated using heat-induced epitope retrieval (HIER) with sodium citrate/EDTA/Tween 20, heat (60C), and pressure (~15 psi) for 72 hr. Cells positive for osterix in construct sections stain brown with 3,3′-diaminobenzidine (DAB; arrows). Cells negative for osterix appear blue with Mayer’s hematoxylin used as a counterstain. Staining intensity of DAB and the number of osterix-positive cells were greatest with a primary antibody dilution of 1:50 (A) compared with other dilutions examined. Scale bar for all panels = 50 µm. Abbreviations: PCL, polycaprolactone; PLLA: poly-l-lactic acid.

After refrigeration, slides were washed (TTBS, 3 × 5 min each) before addition of secondary antibody (ImmPRESS, 30 min; Vector Laboratories, Burlingame, CA) in accordance with directions from the manufacturer. Excess secondary was removed from the slides with three changes of TTBS, 5 min each, followed by a peroxide block (0.3% H₂O₂/30% CH₃OH, 10 min). The peroxide block was rinsed from the slides (TTBS, 3 × 5 min each) before addition of 3,3′-diaminobenzidine (DAB; 1 tablet + 10 mL PBS + 3 drops 3% H₂O₂; MaxTag DAB tablets, Rockland, Limerick, PA) solution. After DAB development, slides were counterstained with Mayer’s hematoxylin (Electron Microscopy Sciences, Hatfield, PA), cleared, mounted with DPX (Electron Microscopy Sciences), and coverslipped for microscopic viewing.

Imaging and Microscopy

Tissue-engineered constructs retrieved 10 weeks following implantation in nude mice were photographed with a camera for gross morphology before fixation. Photomicrographs were obtained under 40× magnification using an inverted light microscope (Model IX70, Olympus, Melville, NY) equipped with a camera (Model CC12; Olympus). Multiple images were collected from the same section area at differing focal points and then “focus-stacked” using Adobe Photoshop (Adobe Systems, Inc., San Jose, CA) auto-blend function to produce a clear picture. Two sets of images from the same area were collected following different parameters. The first set was obtained with the external condenser lens placed in the optical path of the microscope to focus illumination and negate light-refractive qualities of the polymers to provide clear viewing of the cells within specimen sections. In the second set of images, the external condenser lens was removed from the microscope optical path and the images were collected at the same section location. In the absence of the external condenser, electrospun PLLA nanofibers and PCL/PLLA scaffolds were readily identified as a result of their light-refractive capabilities. Where appropriate, pairs of images obtained in the presence or absence of the microscope external condenser lens are presented in the “Results” section to show respectively both cells and polymer constituents of the same specimen sections. Osterix-positive cells appeared brown following DAB staining while any other cells in the section being viewed stained blue with Mayer’s hematoxylin counterstain. Images were recorded and stored using MicroSuite software (version 2.5; Olympus).

Results

Figure 1 compares the staining of constructs in representative sections treated with primary antibody diluted 1:50, 1:100, 1:250, and 1:500 in Bond primary antibody diluent. Counterpart controls using anti-rabbit IgG alone at the same working dilutions were also examined, and an image of a section is shown representing the absence of staining of these constructs at a dilution of 1:50. Staining with a primary antibody dilution of either 1:50 or 1:100 was notably more intense, and the cells were more frequently labeled than staining with dilutions of 1:250 or 1:500. Staining with a dilution of 1:50 yielded a qualitative increase in intensity and cell labeling compared with that of 1:100.

Figure 2 shows the morphology of a typical construct after its retrieval following 10 weeks of implantation in a nude mouse. The surfaces of the construct block, shaped as a rectangular solid, were wrapped with human donor periosteum (Fig. 2A). Bisection of the block revealed the inner PCL/PLLA scaffold core covered with periosteum (Fig. 2B). The PCL/PLLA scaffold core may be coated with very thin layers of electrospun PLLA nanofibers before periosteum is applied and aspects of these and other specimens are represented in more detail in the histological and immunohistochemical images shown in Figures 3–6.

Figure 2.

Optical photographs of a representative intact (A) and bisected (B) construct composed of a core scaffold of PCL/PLLA (75/25) wrapped with human donor periosteum and retrieved after implantation in a nude mouse for 10 weeks. Portions of the surface of the intact specimen are tinged red where vascularization is present from the host mouse. Bisection of the construct shows close integration between the periosteal wrap (P) covering the PCL/PLLA core. Corners of the construct, such as that identified by the arrowhead, show two adjacent aspects of the periosteum which were examined in greater detail to assess the effects of different IHC processing regimes. Scale bar = 0.5 cm. Abbreviations: PCL, polycaprolactone; PLLA: poly-l-lactic acid.

Figure 3.

Light photomicrographs of a corner of an undecalcified PCL/PLLA (75/25) scaffold coated with electrospun PLLA nanofibers, wrapped with human periosteal tissue, and harvested after implantation for 10 weeks. On retrieval, the specimen was fixed in formalin, embedded in paraffin, sectioned and stained. Alizarin red staining (A) of this specimen section highlights significant deposits of calcium present within the periosteal tissue and underlying thin layer of PLLA nanofibers (not distinguishable in the specimen at this magnification). In a section of the specimen consecutive to the first, von Kossa staining (B) demonstrates phosphate deposits (black) in the tissue regions corresponding to those with calcium (A). Areas with an (*) are highlighted in Figs. 5 and 6. Scale bar = 500 µm. Abbreviations: PCL, polycaprolactone; PLLA: poly-l-lactic acid.

Figure 4.

Light photomicrographs of a decalcified PCL/PLLA (75/25) scaffold wrapped with human donor periosteum, implanted for 10 weeks in a nude mouse, fixed in formalin, and sectioned and stained with DAB. In this figure, panel A represents the section image obtained with the external condenser lens placed into the optical path of the microscope and panel B with the lens removed from the path. DAB as an indicator molecule shows osterix as brown within several cells in the image field (A, B; arrows). Counterstaining with Mayer’s hematoxylin indicates cells as blue. The periosteal tissue and electrospun PLLA nanofibers should be present in the upper right portion of the images (A, B), but they separated from the slide and were lost in the retrieval buffer as a result of the high temperature and elevated pressure (120C, ~15 psi above atmospheric, 10 min) used during the epitope retrieval procedure. Despite loss of tissue from slides during processing, portions of the PCL/PLLA scaffold and remnants of electrospun PLLA nanofibers remain and become more clearly visible when the external condenser is removed from the microscope optical path (B). The scaffold and nanofiber appearance is marked by numerous irregular contour lines and fine, thread-like structures, respectively (B). Scale bar = 50 µm. Abbreviations: PCL, polycaprolactone; PLLA: poly-l-lactic acid; DAB, 3,3′-diaminobenzidine.

Figure 5.

Light photomicrographs illustrating a decalcified PCL/PLLA (75/25) scaffold retrieved after 10 weeks of implantation in a nude mouse and corresponding to a region of the lower magnification area in Fig. 3A (*). These sections were treated using HIER under mild conditions (sodium citrate/EDTA/Tween 20, 60C, water bath, ~15 psi, 72 hr). Osterix-positive osteoblasts stained brown with DAB (A, B; arrows) appear within the intact electrospun PLLA nanofiber layer (NF) and underlying PCL/PLLA scaffold of the construct section. In relation to the periosteum (P) overlying the construct, the NF layer and PCL/PLLA scaffold are clearly defined by irregular contours and thread-like structures in the absence of the external condenser lens from the optical path of the microscope (B). The tissue section was counterstained using Mayer’s hematoxylin. Light photomicrographs of a negative control (C, D; no osterix antibody addition) IHC specimen from constructs HIER-treated under mild conditions as above for a construct shown in (A, B). Cells stained with Mayer’s hematoxylin (blue) are visible within the periosteum (P), electrospun PLLA nanofiber layers (NF), and underlying PCL/PLLA scaffold (PCL/PLLA) (C, D). No osterix-positive cells are apparent. Structural damage to the P, NF, and PCL/PLLA is minimal in comparison with the results presented in Fig. 4. Little background staining of DAB is visible in the polymers or tissue. Scale bar = 50 µm. Abbreviations: PCL, polycaprolactone; PLLA: poly-l-lactic acid; HIER, heat-induced epitope retrieval.

Figure 6.

Light photomicrographs of osterix-positive cells in two different areas enlarged from the representative specimen shown in Fig. 3A (**). Many such brown-stained cells (arrows) are present within the construct (A) and its electrospun PLLA nanofiber layers (NF) marked by fine, thread-like structures (B) made visible in the absence of the external light condenser from the microscope optical path. The thin PLLA fibers are randomly dispersed and approximately 1 µm in diameter (B). Osteoblasts both osterix-positive or osterix-negative (stained blue with Mayer’s hematoxylin) have migrated into the NF layer from the overlying wrapped periosteum (P) (A, B). In another region of the same specimen, cells are present in linear fashion (arrows) along the interface between NF and PCL/PLLA areas (C, D). In this instance and where the absence of the external condenser lens from the optical path of the microscope reveals PLLA nanofibers and the substructure of PCL/PLLA (D), the cells have traversed the nanofiber layers (NF). Many cells here are positive for osterix (D) and all such osterix-positive cells (A–D) correlate with histological staining of mineral depicted in Fig. 3. Some background staining of DAB is present in the PCL/PLLA scaffold. Scale bar = 50 µm. Abbreviations: PCL, polycaprolactone; PLLA: poly-l-lactic acid.

Figure 3 depicts representative histological images of a corner of an undecalcified construct consisting of a PCL/PLLA scaffold coated with electrospun PLLA nanofibers, wrapped with human periosteal tissue, and harvested after 10 weeks of implantation in vivo. Calcium stained with alizarin red was evident at the interface between the periosteal wrap and the PCL/PLLA inner scaffold of this construct (Fig. 3A). In a similar manner, phosphate stained black by von Kossa treatment (Fig. 3B) mimicked the area stained with alizarin red. Taken together from consecutive construct sections, the pattern of alizarin red and von Kossa staining indicated the presence of mineral (apatite crystals) in these specimens.

Table 1 shows results from PIER methods applied to decalcified samples using pronase, type 1 collagenase, or mixtures of both. All such approaches were ineffective. Tissue specimens suffered no apparent structural damage, but intracellular staining of DAB was not observed in any of the specimens treated using PIER methods (data not shown). Mayer’s hematoxylin counterstain highlighted cells within the periosteum, layers of PLLA nanofibers, and underlying PCL/PLLA scaffold (data not shown).

HIER methods were initially unsuccessful in all buffers when a water bath was used as the heat source (65–95C) for short periods of time (<48 hr, Table 1). No labeling of osterix was visible within any of the cells of samples examined (data not shown). Damage occurred to tissue sections treated with sodium citrate/EDTA buffer containing sodium dodecyl sulfate and resulted in complete delamination of sections from the slides (Table 1).

Positive results for immunostaining of osterix in this series of experiments were achieved using sodium citrate/EDTA/Tween 20 buffer as the epitope retrieval solution (Table 1). Figure 4A presents a composite of 16 focus-stacked light micrograph images and illustrates positive staining of osterix present within osteoblasts in a representative decalcified construct following high-temperature, elevated-pressure epitope retrieval (120C, 30 psi, 10 min) using an autoclave. Periosteal tissue, electrospun PLLA nanofibers, and portions of the PCL/PLLA scaffold delaminated from the slides during these epitope retrieval conditions and were lost in the buffer solution in this instance.

Some regions of the PCL/PLLA scaffolds remained on slides (Fig. 4A and B), but the identification and localization of additional osterix-positive cells with parts of the sections missing could not be definitely determined within the constructs as a whole.

Positive IHC immunostaining results for osterix on another slide of the representative construct in Fig. 3 are shown in Fig. 5A and B. Osterix-positive cells were present within the periosteum, the electrospun PLLA nanofibers, and the PCL/PLLA scaffold (arrows) following epitope retrieval conditions at low temperature and normal pressure with extended time (60C, ~15 psi, 72 hr; sodium citrate/EDTA/Tween 20 buffer). Unlike constructs similar to that shown in Fig. 4, the electrospun PLLA nanofibers and PCL/PLLA scaffold exhibited no apparent structural damage under these circumstances (Fig. 5A and B).

Figure 5C and D are light photomicrographs of a representative negative control slide from a construct also processed following mild parameters and longer time for epitope retrieval (60C, ~15 psi, 72 hr; sodium citrate/EDTA/Tween 20 buffer). While no osterix-positive cells were observed, Mayer’s hematoxylin counterstain showed cells within periosteal tissue, the electrospun PLLA nanofibers, and the PCL/PLLA scaffold (Fig. 5C and D).

Figure 6A and B are light photomicrographs illustrating the presence of osterix-positive cells (arrows) clearly located in the electrospun PLLA nanofibrous layers in a different area along the periosteal-scaffold border represented by the asterisk pair in Fig. 3.

The PLLA nanofibers appeared as thin, intermingled individual strands as deposited by electrospinning on the underlying PCL/PLLA scaffold (Fig. 5B). Brown-stained osterix-positive cells had infiltrated the nanofibers from the periosteal layer. In Fig. 6C and D, cells were found in linear arrangement along the interface between nanofibers and PCL/PLLA components of the specimen. Periosteal tissue, layers of electrospun PLLA nanofibers, and the underlying PCL/PLLA scaffold were evident in the absence of the external condenser lens from the microscope optical path (Fig. 6D). The morphology of osteoblasts in linear disposition present at the nanofiber–PCL/PLLA scaffold interface (Fig. 6C and D, arrows) correlated with mineral formation observed in Fig. 3. Minor nonspecific background staining of DAB was present within the PCL/PLLA scaffold (Fig. 6C and D).

Discussion

This article describes the successful labeling of osterix protein in cells from human donor periosteum used to wrap scaffolds of interest to the field of tissue engineering, in this instance PCL/PLLA scaffolds coated with electrospun PLLA nanofibers. Osterix (SP7, Osx) is an osteoblast-specific zinc-finger transcription factor that is critical to the development and production of bone tissue.^27–30 Nakashima et al.²⁷ showed that inactivation of the Osx gene in mice resulted in little to no bone formation. In locations where bones were supposed to grow and mineralize in normal mice, there were instead groups of undifferentiated mesenchymal cells and tissue in Osx-null mice. The only locations with mineral deposition and the potential for bone growth resulted from the presence of chondrocytes that provided a template for osteoblast recruitment and bone matrix formation.²⁷ In its role as a principal factor in the elaboration of bone tissue, osterix helps with regulation of other proteins crucial for osteoblast differentiation. These include bone morphogenetic protein 6,²⁹ calmodulin-dependent kinase II,³⁰ Wnt/β-catenin,^31,32 and Runx2.³³ All of these molecules are involved in the development of osteoblasts and maintenance of mature bone tissue although Runx2 is also associated with the development of cells having chondrogenic potential.^34,35 For preosteoblasts, which hold potential for either chondrogenic or osteogenic pathways, osterix directs osteoblast, rather than chondroblast, development and maturation.³⁶

Osterix is an intracellular protein detected primarily within the nucleus of only preosteoblasts and osteoblasts. Osterix does not appear to any measurable extent in the extracellular spaces of bone tissue and would therefore be a critical marker in determining not only the regenerative potential of tissue-engineered constructs but also the location and migration of osteoblast-specific cells. In the experiments here, human donor periosteal tissue was removed from its native bone and wrapped around synthetic polymer scaffolds. After 10 weeks of implantation of the resulting constructs in athymic mice, a measure of whether the donor periosteum retained its biological activity and osteogenic potential during this time interval was determined by the presence and location of osterix in the periosteal cells. In this regard, identification and localization of human osterix in the periosteum and other regions of such tissue-engineered constructs were achieved with immunohistochemical labeling using the protocol detailed above.

The successful procedure was modified from IHC techniques designed for labeling proteins in soft tissues rather than tissue such as periosteum, giving rise to mineralized extracellular matrices. A few steps included in this modified IHC protocol are not required for soft, unmineralized specimens, and it is useful to point out differences in the two approaches. For the modified methodology, first, the slides holding sections of specimens wrapped with periosteum were immersed in 10% NBF overnight before the epitope retrieval process to limit potential structural damage of the tissue incurred during decalcification. Second, a decalcification process, using the formic acid–based decalcification agent, Immunocal, was critical for removing residual calcium and phosphate possibly present in the tissue sections that could interfere with binding of the osterix antibody as well as sectioning. Regarding decalcification additionally, this procedure obviated the possible need for methyl methacrylate embedding, typically used for successful sectioning of undecalcified specimens. To minimize tearing and related artifacts in the sectioning of the paraffin-embedded samples examined in the present study, the blocks of cell-scaffold constructs were placed overnight in a −80C freezer before microtomy and maintained cold as they were cut. The low temperature of the block face was helpful to the collection of intact sections. In another related context, it should be also noted that long fixation times for constructs (a week in formalin in this study) did not interfere with tissue and polymer integrity and subsequent IHC processing. Mineralized samples and polymeric materials are known to respond advantageously to such fixation regimes, and antibodies are likewise minimally affected.^23,24

Following the decalcification steps, a series of routine epitope retrieval protocols was used in an effort to find a suitable approach to labeling osterix within the tissue specimens. HIER using varying buffers and temperatures and PIER with different proteases were examined for their efficacy in revealing osterix protein for labeling while producing minimal damage to the tissue samples. The particular specimen constructs investigated here resulted in a degree of difficulty in IHC processing as they consisted of both tissue and the synthetic PCL and PLLA polymers or copolymers. Polymeric materials were more susceptible than tissue to delamination and degradation induced by heating methods and solution parameters in the IHC process. In addition, protein blocking was altered for these experiments to minimize the chances of false-positive results as a consequence of possible secondary antibody binding. In place of the horse serum provided by the manufacturer of the ImmPRESS polymer kit, Background Buster, a universal peptide blocker developed for use with human and animal tissues, was applied to limit background staining as a result of any secondary antibody binding to the charged polymers or murine tissue in these specimens.

For protease-induced retrieval methods, no major changes were made to established methodology.^14,15,22 The tissue specimens treated with type 1 collagenase and/or pronase and placed in a humidified chamber at 37C did not exhibit visible damage to the structure of the tissue or polymer scaffold. However, attempts to use PIER methodologies to label and reveal the osterix epitope were unsuccessful in this series of experiments. With consideration that increased enzymatic digestion can also cause tissue damage, the potential exists to extend the time for protease retrieval methods and other proteases (proteinase k and pepsin, for example) could be used. Nonetheless, heat-induced retrieval methodology demonstrated initial success in revealing the epitope for the osterix antibody and was selected for further investigation.

Heat-induced retrieval methods resulted in greater variability with respect to tissue damage and structural integrity of the polymer scaffolds. In the instances in which sodium citrate/SDS was used as the epitope retrieval buffer, sections delaminated from slides entirely, likely a consequence of SDS as a strong, anionic surfactant.³⁷ For slides treated with Tris-HCl, only half retained sections through the remainder of each protocol and all such slides were absent of any visible labeling of osterix. For sodium citrate/EDTA/Tween 20 buffer, conditions for successful epitope retrieval were achieved initially with slides in the buffer solution in an autoclave at 120C and 15 psi for 10 min. The high temperature and pressure, used in a number of routine retrieval protocols,^16–18 caused complications as the electrospun PLLA nanofibers and PCL/PLLA scaffold were removed partially and the periosteum was removed entirely from slides. In these procedures, determining the precise location of osterix-positive cells in the overall structure of the constructs was not possible. On the other hand, osterix labeling present in autoclaved specimens suggested that heating was an appropriate parameter for retrieving the osterix epitope.

Based on the results of a series of primary antibody dilutions as detailed above, a dilution of 1:50 was determined for application in the investigation. This dilution validated the recommendation of the manufacturer as an optimal value for detecting osterix in the specimens of interest in the present work. Counterpart controls of anti-rabbit IgG alone in place of the primary antibody at the same working dilutions yielded no staining of constructs as expected.

Obtaining clear images of autoclaved slides using light microscopy was frequently challenging. Portions of tissue sections, for example, no longer lay flat on the surface of slides as a result of section delamination induced by the high heat and pressure of the retrieval process. Consequently, multiple microscopic images were recorded at varying focal points, collected and focus-stacked to generate a single, clear image of the tissue. While overcoming the problem of imaging specimen areas in slightly different focal planes, this technique was otherwise somewhat limited as it used a three-dimensional set of images projected onto a single, two-dimensional plane. In this case, the potential exists for overlapping areas of tissue to distort a final image and lead to possible uncertain interpretation. Despite the latter consideration, these specific concerns regarding tissue integrity and clear imaging ultimately produced successful epitope retrieval achieved at decreased temperature for increased time to identify and localize osterix conclusively in cells of the tissue-engineered construct samples.

The epitope retrieval process is directed toward removing the crosslinks generated between and within tissue specimens during the formalin-fixation process. Heat-induced retrieval is also associated with denaturation and refolding of proteins of interest.^4,14,22 The denaturation and refolding process is time- and temperature-dependent. High temperature such as the 120C reached in an autoclave here decreased the time required for protein conformation to return to a state in which the antibody recognized its target sequence of amino acids. Reduction of the target temperature achieved during the retrieval process to preserve tissue and polymer scaffold morphology required a significant extension of the time period of heating. Specifically, epitope retrieval using sodium citrate/EDTA/Tween 20 buffer for 72 hr at 60C yielded successful labeling of osterix protein and maintained structural integrity of the tissue in the process.

It is difficult to improve IHC procedures for proteins that are found in specimens also containing synthetic polymer materials as well as mineral. With the field of tissue engineering expanding to new avenues in biology, medicine and other areas, developing histochemical methods to study living tissue, synthetic materials, and mineral is important for furthering the landscape of regenerative medicine. Epitope retrieval is a critical step in immunohistochemical techniques applied to fixed and embedded samples or those that may be completely untreated. A balance between unmasking of epitopes of a protein of interest and preservation of the structure of the tissue specimen must be established to produce high-quality IHC staining results. Heat was shown to be a critical component in retrieval of the osterix epitope for the tissue samples presented in this work, but extreme autoclave heat and pressure during the retrieval process altered the structure of the original tissue to the degree that it was not possible to identify osterix or determine the exact location of osterix-positive cells in tissue sections. Heat applied at a lower temperature during epitope retrieval and for a limited time preserved the original tissue structure of specimens while successfully retrieving the osterix epitope. Parenthetically, this methodology applied to other antibodies in this laboratory has yielded very satisfactory results. The approach, then, has wider application than that described here for osterix and circumvents the need to develop epitope retrieval methods for additional antibodies being investigated.

An increased epitope retrieval time necessary to complete the osterix staining protocol outlined in the preceding pages of this article is significant. It points to the need to examine all aspects of the IHC process to develop protocols to tag a protein without significantly impacting original tissue structure. Apparently simple modifications to established protocols, such as increasing the amount of time during epitope retrieval, may be important factors in whether an IHC technique succeeds in labeling a target protein. This study clearly shows the advantages of using the methodology described to demonstrate for the first time that cells considered to be osteoblasts may be confirmed as such by IHC through positive staining of osterix, an osteoblast-specific transcription factor. In addition, the work presents novel findings that osteoblasts from periosteum migrate into and through a nanofiber layer electrospun over a tissue-engineered scaffold in the constructs designed here. IHC continues to be a versatile tool for identifying and localizing proteins critical for characterizing the nature of cells and tissues in studies in many basic science and clinical research fields.